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Virulence for chickens of Clostridium perfringens isolated from poultry and other sources
Anaerobe 16 (2010) 289e292
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Pathogenesis and Toxins
Virulence for chickens of Clostridium perfringens isolated from poultry and other sources Kerry K. Cooper, James R. Theoret, Bernard A. Stewart, Hien T. Trinh, Robert D. Glock, J. Glenn Songer* Department of Veterinary Science and Microbiology, University of Arizona, 1117 E. Lowell St., Tucson, AZ 85721, USA
a r t i c l e i n f o
a b s t r a c t
Article history: Received 20 September 2009 Received in revised form 17 February 2010 Accepted 21 February 2010 Available online 1 March 2010
Clostridium perfringens type A is the most common cause of poultry necrotic enteritis (NE). Of the four “major” toxins, type A strains produce only alpha toxin (CPA), which has long been considered a major factor in pathogenesis of NE. We investigated the virulence for poultry of type A strains from a variety of enteric sources. Newly-hatched Cornish Rock chicks were fed a low protein diet for one week, a high protein diet for a second week, and then challenged with log-phase cultures of C. perfringens, mixed 3:4 (v/v) with high protein feed. Strain JGS4143 [genotype A, beta2 positive (cpb2pos), from a field case of NE] produced gross lesions compatible with NE in >85% of challenged birds. However, strains JGS1714 (enterotoxigenic genotype A, cpb2pos, human food poisoning), JGS1936 (genotype A, cpb2neg, bovine neonatal enteritis), JGS4142 (genotype A, cpb2pos, bovine jejunal hemorrhage syndrome), JGS1473 (genotype A, cpb2pos, chicken normal flora), JGS1070 (genotype C, cpb2pos, porcine hemorrhagic enteritis), JGS1882 (genotype A, cpb2pos, porcine neonatal enteritis), JGS1120 (ATCC 13124, genotype A, cpb2neg, gas gangrene), JGS4151 (strain 13, genotype A, cpb2pos, canine), and JGS4303 (SM101, enterotoxigenic genotype A, cpb2neg, human food poisoning) failed to produce disease. In vivo passage failed to increase virulence of the non-NE strains. NE strains must have specific poultry-associated virulence attributes, such as the recently identified NetB and other factors, which allow for the development of disease. Ó 2010 Elsevier Ltd. All rights reserved.
Keywords: Clostridium perfringens Poultry Necrotic enteritis Broiler chickens Virulence
1. Introduction Poultry necrotic enteritis (NE) is most commonly caused by Clostridium perfringens type A [1e5], although occasional cases are due to type C infection [6e10]. This species is anaerobic, sporeforming, gram-positive, and rod-shaped, and is divided into five toxinogenic types based on the pattern of production of four major toxins [4,11]. Control of NE has been most commonly achieved by use of antimicrobials [3]. However, concerns over downstream human health effects, such as resistance transfer from animal pathogens to human pathogens, have led to European Union bans on use of antimicrobial growth promoters [12e15]. A further increase in incidence of NE has also resulted from the introduction of products for immunoprophylaxis of coccidiosis [16,17]. Attenuated coccidia in vaccines may produce transient damage to intestinal epithelia, facilitating establishment of C. perfringens and production of NE [5,15,18,19]. A more important factor may be cessation of the use of
* Corresponding author. Tel.: þ1 520 621 2962; fax: þ1 520 621 6366. E-mail address: [email protected] (J.G. Songer). 1075-9964/$ e see front matter Ó 2010 Elsevier Ltd. All rights reserved. doi:10.1016/j.anaerobe.2010.02.006
ionophore coccidiostats. These compounds have been extraordinarily effective in preventing losses due to infection by Eimeria spp, but have also provided serendipitous anti-clostridial effects [15,20e23]. Consequently, NE has re-emerged as a major problem for the poultry industry [5,12,20,24,25]. Alpha toxin (CPA) has long been considered a critical virulence factor in the pathogenesis of NE, although specific evidence has been lacking. CPA is the only “major” toxin produced by type A strains, and higher levels have been detected in birds with NE than in normal birds [4,26]. Oral challenge of germ-free chicks with crude toxin preparations has resulted in disease development, and these effects were neutralized by anti-CPA serum mixed with the crude toxin prior to challenge [3,27]. Additionally, birds recovering from NE have high levels of anti-CPA antibodies, whereas healthy birds have low to undetectable levels [26,28]. Several studies have found that vaccinating birds with CPA provides partial protection against NE [29e31]. The role of CPA has recently been called into question. Vaccination with a CPA-deficient mutant protected against experimental challenge [32]. Seemingly more conclusive evidence was provided by the finding that an engineered CPA mutant of a virulent NE strain produced disease [33].
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A vital element in the development of prevention and control measures for NE is a better understanding of pathogenesis. We developed an experimental model of C. perfringens type A infection; strains isolated from field cases of NE are virulent, although variably so (unpublished data). Earlier, we reported results of preliminary work, demonstrating that virulence of C. perfringens type A for chickens is strain specific [34,35] and formalize those findings here. The overall objective of this work was to determine if CPAproducing type A strains from a variety of sources are virulent for chickens in our experimental model. Type A strains from normal chickens or from enteric disease in other species do not produce NE in chicks. This adds evidence to the argument that the virulence armada of NE strains includes factors other than CPA, including in many cases NetB [36] and other factors (unpublished data). 2. Methods and materials 2.1. Birds and care Commercial Jumbo Cornish Rock broiler chicks were obtained as day-old hatchlings from Murray McMurray Hatchery (Webster City, IA, USA). Birds were housed in 1.5 m diameter brooders constructed of 3 mm thick pegboard and divided into three equal parts. Commercial wood shavings provided as bedding were changed at two week intervals. Birds were fed a commercial chick starter ration (20% protein, Eagle Milling, Casa Grande, AZ, USA) through experimental day 7. Thereafter, birds were fed a high protein feed (28% protein) mixed 50:50 with menhaden fishmeal (SeaLac, Omega Protein, Houston, TX, USA). On day 14, feed was withheld for 20 h, and birds were then challenged with C. perfringens. Water was available ab lib in galvanized steel automatic waterers throughout each study. 2.2. Challenge inoculum and protocol Strains used in these studies are listed in Table 1, with host and disease (if any) of origin. Strains were stored at 80 C in 50% glycerol:50% brain heart infusion (BHI; Difco, Detroit, MI, USA). For inoculum preparation, approximately 10 ml were streaked for isolation on a plate of BHI agar with 5% citrated bovine blood. After incubation under anaerobic conditions (5% H2: 5% CO2: 90% N2) at 37 C for 24 h, 1e2 colonies were transferred into 10 ml of cooked meat medium (CMM, Difco) in Hungate tubes and incubated in the same atmosphere at 37 C for 18 h. The resulting culture was used to inoculate 100 ml fluid thioglycollate broth (FTG, Difco), which, after incubation as before, was diluted 1:10 in CMM and incubated as before. Thirty-three millilitre of the CMM culture were used as inoculum for 1 l FTG medium, and after 18 h incubation, this was mixed with feed for challenge. A separate serially-passed culture was prepared for each challenge feeding (total n ¼ 8). Numbers of Table 1 Clostridium perfringens strains. Genotype
Source of strains
netB
JGS1070 JGS1120 JGS1473 JGS1714 JGS1882 JGS1936 JGS4303 JGS4142 JGS4143 JGS4151
C, cpb2þ A A, cpb2þ A, cpeþ A, cpb2þ A A, cpeþ A, cpb2þ A, cpb2þ A, cpb2þ
Porcine hemorrhagic enteritis ATCC 13124a Avian normal flora Human foodborne disease Porcine neonatal enteritis Bovine neonatal enteritis SM101a Bovine jejunal hemorrhagic syndrome Field case NE Strain 13b
Neg. Neg. Neg. Neg. Neg. Neg. Neg. Neg. Pos. Neg.
a
Sequenced (Ref. [35]). Sequenced (Ref. [41]).
2.3. PCR-C. perfringens genotyping [37] Isolated colonies on BHI agar with 5% citrated bovine blood were suspended in 200 ml of HPLC-grade water (Mallinckrodt, Hazelwood, MO, USA), boiled for 20 min, and centrifuged 13,000 g for 2 min. The supernatant fluid served as template DNA, and 5 ml were added to 20 ml of a master mix solution containing primers, Taq polymerase (Promega, Madison, WI, USA), MgCl2 (25 mM, Sigma-Aldrich), dNTPs (5 mM, Promega), and 10 Assay Buffer (Promega), to give a total reaction volume of 25 ml. PCR amplification was comprised of 35 cycles of denaturing (94 C, 1 min), primer annealing (55 C, 1 min) and extension (72 C, 1 min). Aliquots (15 ml) of each PCR product were loaded into wells of a 1.5% (w/v) agarose gel (GenePure LE, ISC BioExpress, Kaysville, UT, USA), stained with 0.01% (v/v) ethidium bromide (Sigma-Aldrich), and separated by electrophoresis. PCR products were visualized via UV transillumination and digitally photographed. 2.4. Pulsed-field gel electrophoresis (PFGE) Genotyped strains were embedded in plugs of 2% CleanCut agarose (Bio-Rad, Hercules, CA, USA), according to the method of Lin and Labbe [38]. After cell lysis and DNA digestion with SmaI (New England Biolabs, Ipswich, MA, USA), electrophoresis was performed on a CHEF Mapper DRII (Bio-Rad) at 6.0 V/cm for 20 h with an angle of 120 , an initial pulse time of 0.50 s, and a final pulse time of 40 s. After staining with ethidium bromide, gels were examined and photographed via UV transillumination. 2.5. Assays of alpha toxin (CPA; lecithinase) activity [39]
JGS #
b
colony-forming units (cfu) were determined by plating serial 10fold dilutions on BHI agar. Birds were inoculated on days 15e18. High protein feed and FTG medium culture were mixed in a ratio of 3:4 (v/v). The mixture, which had a paste-like consistency, was then placed in galvanized steel feed trays and offered to chicks. Trays were cleaned and remaining feed disposed of prior to each subsequent feeding. Negative control birds were challenged with un-inoculated FTG mixed with high protein feed at the same ratio. On day 19, birds were euthanized by CO2 asphyxiation. Necrotic intestinal lesions were scored (0: no gross lesions; 1þ: thin-walled or friable small intestine; 2þ: focal necrosis or ulceration; 3þ: large patches of necrosis; 4þ: severe or extensive necrosis typical of field cases) and segments fixed in 10% buffered formalin or retained fresh for bacteriological culture. Intestinal contents serially diluted in phosphate buffered saline [PBS; 0.01 M, pH 7.2] were plated on C. perfringens selective agar (CPSA [BHI agar supplemented with 0.5% yeast extract (Becton Dickinson, Franklin Lakes, NJ, USA), 0.1% sodium metabisulfite (Sigma-Aldrich, St. Louis, MO, USA), 5% citrated bovine blood, and D-cycloserine (400 mg/ml; Sigma-Aldrich)] and incubated under anaerobic conditions (as above) for 24 h.
Egg yolk emulsion was prepared by adding one asepticallyremoved egg yolk to 20 ml DMG buffer [0.04 M 3,3 dimethylglutaric acid (Sigma-Aldrich) pH 7.0 plus 0.008 M CaCl2 (Sigma-Aldrich), 0.0002 M ZnSO4 (Sigma-Aldrich), and 0.1% bovine serum albumin (Sigma-Aldrich); pH adjusted to 7.2 and volume adjusted to 100 ml with HPLC-grade water] and mixed thoroughly and centrifuged (26,890 g, 20 C, 20 min). Supernatant was diluted 1:10 in DMG buffer prior to use. Two-fold serial dilutions (1:2e1:256) of purified recombinant CPA or test samples were prepared in DMG buffer, and 100 ml of each were added to wells of a 96-well plate. An aliquot (100 ml) of egg yolk emulsion was then
K.K. Cooper et al. / Anaerobe 16 (2010) 289e292
added to each well and the plate sealed to prevent evaporation. After incubation at 37 C for 1 h, results were read spectrophotometrically at 450 nm. Amounts of CPA in test samples were derived by comparison of results to a plot generated by testing purified recombinant CPA standards. 2.6. Institutional animal care and use committee (IACUC) approval Studies presented here were pre-reviewed and pre-approved by the University of Arizona IACUC, under protocol number 02-204. 3. Results We examined the ability of isolates of C. perfringens (Table 1) to produce NE in our model. Disease did not result from challenge with these isolates, although 58/66 (87.9%) positive controls (inoculated with NE isolate JGS4143) developed gross lesions (Fig. 1) with an average lesion score of 2.31 (Table 2, Fig. 2). Negative controls (challenged with un-inoculated thioglycollate broth) did not develop lesions. There were no statistically significant differences in CPA production across strains and, in fact, positive control strain JGS4143 produced less CPA than any other strain in vitro (Table 2). Intestinal samples from challenged birds were examined by bacteriologic culture for C. perfringens, and PCR-genotyped and recovered isolates were confirmed as the challenge strain in each case by comparing PFGE profiles. Attempts to recover challenge strains in initial trials were successful only from birds inoculated with strains JGS4143 and JGS1473 (avian normal flora). Further groups of birds were challenged with JGS1070, JGS1714, JGS1882, JGS1936, and JGS4142, as well as strain JGS4151 [strain 13 [40]]. No challenged birds developed lesions, but PFGE analysis of at least one-half of the isolates (up to 100 per bird) showed that we recovered all challenge isolates except JGS1070 (a type C porcine isolate). Only avian isolates (JGS4143 and JGS1473) were isolated from the jejunum, while JGS1714 was only recovered from the duodenum of challenged birds. The other non-avian isolates (JGS1882, JGS1936, JGS4142, JGS4151) were isolated from the ileum of challenged birds. Birds were then inoculated with these isolates (JGS1882, JGS1714, JGS1936, JGS4142, and JGS4151) and JGS1473, all after passage through chicks, to determine if in vivo passage led to detectable virulence. Again, no challenged birds developed lesions. Positive control birds challenged with JGS4143 developed gross lesions [15/16 (93.7%), average lesion score 3.07] and negative controls did not.
Fig. 1. Macroscopic examination of the intestine in birds challenged with C. perfringens. A. Gross lesions typical of necrotic enteritis from bird challenged with JGS4143. B. Lack of gross lesions in intestine of bird challenged with JGS4142; this is typical of intestines of birds challenged with non-NE strains.
291
Table 2 Response of birds to challenge with non-avian strains of Clostridium perfringens. Strain
Birds with gross lesion/total (%)
Average lesion score SD
Average challenge inocula titer (log[10]) SD
Average challenge inocula CPA titer (mg/ml) SD
JGS1473 JGS1070 JGS1882 JGS4142 JGS1714 JGS1936 SM101 Strain 13 ATCC 13124 JGS4143 Negative control
0/16 (0.0%) 0/16 (0.0%) 0/16 (0.0%) 0/15 (0.0%) 0/16 (0.0%) 0/16 (0.0%) 0/18 (0.0%) 0/20 (0.0%) 0/18 (0.0%) 58/66 (87.9%) 0/67 (0.0%)
0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 2.31 0.50 0.0
6.98 1.6 5.70 0.41 5.86 0.99 5.88 0.88 5.88 0.87 8.60 0.47 N/A N/A N/A 8.48 0.65 N/A
884.33 42.52 903.63 83.69 921.58 40.2 923.25 74.57 943.33 46.25 932.92 59.14 N/A N/A N/A 844.42 33.27 N/A
4. Discussion The results of these studies confirm our earlier findings [34,35] and those of others [41] that not all type A strains of C. perfringens produce NE in birds under the challenge conditions used. In fact, we have shown that in vivo passage of NE strains can dramatically increase their virulence (unpublished), but in these studies, a single in vivo passage apparently did not alter virulence. All avirulent
Fig. 2. Microscopic examination of the intestine in birds challenged with C. perfringens. A. Intestine of bird challenged with JGS4143. Extensive fibrinonecrotic inflammation, with bacteria and distended crypts and inflammatory infiltrate in the lamina propria (40). B. Intestine of bird challenged with JGS1473. No microscopic lesions are observed. Findings typical of intestines from birds challenged with other non-NE strains (40).
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strains produced CPA, and none produced NetB [36], suggesting that CPA is not the primary virulence attribute. In addition, these results and the finding that inoculation of birds with CPAcontaining culture supernatant fluid did not produce gross lesions (unpublished data), led us to conclude that the quantity of CPA in experimental inocula does not significantly affect the ability of an isolate to produce disease. Only JGS4143 and JGS1473 were recovered from jejunum, the most common site of lesion development. JGS1882, JGS1936, JGS4142, and JGS4151 were isolated from the ileum post-challenge, which is in keeping with findings of others that C. perfringens most commonly colonizes the ileum and the ceca of chickens as part of the normal flora [42]. JGS1714 was isolated from the duodenum, but not the jejunum or the ileum, suggesting possible lack of colonization factors that might allow avian strains to colonize the jejunum. Additionally, the failure of non-avian strains to effectively colonize birds following challenge with high levels of inoculum also suggest an overall deficiency in poultry colonization factors. In conclusion, NE strains must have specific poultry-associated virulence attributes, such as the recently identified NetB and other factors, which allow for the development of disease. Additionally, failure of sequenced strains (Table 2) to produce disease suggests that they will provide an excellent background for comparisons with genome sequences of NE strains, possibly allowing identification of additional specific virulence factors. Acknowledgements
[15]
[16] [17]
[18] [19]
[20]
[21]
[22]
[23]
[24]
[25]
[26] [27]
The authors thank Dr. Sharon Dial for assistance with the histology from the studies, and Drs. Lynn Joens and Michael Riggs for helpful commentary. This research was funded in part by a grant from Schering-Plough Animal Health. References [1] Al-Sheikhly F, Truscott RB. The interaction of Clostridium perfringens and its toxins in the production of necrotic enteritis of chickens. Avian Dis 1977;21:256e63. [2] Lovland A, Kaldhusdal M. Liver lesions seen at slaughter as an indicator of necrotic enteritis in broiler flocks. FEMS Immunol Med Microbiol 1999;24:345e51. [3] Lovland A, Kaldhusdal M, Redhead K, Skjerve E, Lillehaug A. Maternal vaccination against subclinical necrotic enteritis in broilers. Avian Pathol 2004;33:83e92. [4] Songer JG. Clostridial enteric diseases of domestic animals. Clin Microbiol Rev 1996;9:216e34. [5] Williams RB, Marshall RN, La Ragione RM, Catchpole J. A new method for the experimental production of necrotic enteritis and its use for studies on the relationships between necrotic enteritis, coccidiosis and anti-coccidial vaccination of chickens. Parasitol Res 2003;90:19e26. [6] Gardiner MR. Clostridial infections in poultry in Western Australia. Aust Vet J 1967;43:359e60. [7] Kaldhusdal M, Hofshagen M, Lovland A, Langstrand H, Redhead K. Necrotic enteritis challenge models with broiler chickens raised on litter: evaluation of preconditions, Clostridium perfringens strains and outcome variables. FEMS Immunol Med Microbiol 1999;24:337e43. [8] Kaldhusdal M, Schneitz C, Hofshagen M, Skjerve E. Reduced incidence of Clostridium perfringens-associated lesions and improved performance in broiler chickens treated with normal intestinal bacteria from adult fowl. Avian Dis 2001;45:149e56. [9] Kwatra MS, Chaudhury B. A presumptive diagnosis in the fowl (Gallus gallus domesticus) from Assam (India). Avian Dis 1976;20:401e6. [10] Prescott JF, Sivendra R, Barnum DA. The use of bacitracin in the prevention and treatment of experimentally-induced necrotic enteritis in the chicken. Can Vet J 1978;19:181e3. [11] Nauerby B, Pedersen K, Madsen M. Analysis by pulsed-field gel electrophoresis of the genetic diversity among Clostridium perfringens isolates from chickens. Vet Microbiol 2003;94:257e66. [12] Casewell M, Friis C, Marco E, McMullin P, Phillips I. The European ban on growth-promoting antibiotics and emerging consequences for human and animal health. J Antimicrob Chemother 2003;52:159e61. [13] Emborg H, Ersboll AK, Heuer OE, Wegener HC. The effect of discontinuing the use of antimicrobial growth promoters on the productivity in the Danish broiler production. Prev Vet Med 2001;50:53e70. [14] Emborg HD, Andersen JS, Seyfarth AM, Wegener HC. Relations between the consumption of antimicrobial growth promoters and the occurrence of
[28] [29]
[30]
[31]
[32]
[33]
[34]
[35]
[36]
[37] [38]
[39]
[40]
[41]
[42]
resistance among Enterococcus faecium isolated from broilers. Epidemiol Infect 2004;132:95e105. Van Immerseel F, De Buck J, Pasmans F, Huyghebaert G, Haesebrouck F, Ducatelle R. Clostridium perfringens in poultry: an emerging threat for animal and public health. Avian Pathol 2004;33:537e49. Weber FH, Evans NA. Immunization of broiler chicks by in ovo injection of Eimeria tenella sporozoites, sporocysts, or oocysts. Poult Sci 2003;82:1701e7. Weber FH, Genteman KC, LeMay MA, Lewis Sr DO, Evans NA. Immunization of broiler chicks by in ovo injection of infective stages of Eimeria. Poult Sci 2004;83:392e9. Williams RB. Anticoccidial vaccines for broiler chickens: pathways to success. Avian Pathol 2002;31:317e53. Martel A, Devriese LA, Cauwerts K, De Gussem K, Decostere A, Haesebrouck F. Susceptibility of Clostridium perfringens strains from broiler chickens to antibiotics and anticoccidials. Avian Pathol 2004;33:3e7. Grave K, Kaldhusdal MC, Kruse H, Harr LM, Flatlandsmo K. What has happened in Norway after the ban of avoparcin? Consumption of antimicrobials by poultry. Prev Vet Med 2004;62:59e72. Brennan J, Bagg R, Barnum D, Wilson J, Dick P. Efficacy of narasin in the prevention of necrotic enteritis in broiler chickens. Avian Dis 2001;45: 210e4. Elwinger K, Schneitz C, Berndtson E, Fossum O, Teglof B, Engstom B. Factors affecting the incidence of necrotic enteritis, caecal carriage of Clostridium perfringens and bird performance in broiler chicks. Acta Vet Scand 1992;33:369e78. Watkins KL, Shryock TR, Dearth RN, Saif YM. In-vitro antimicrobial susceptibility of Clostridium perfringens from commercial turkey and broiler chicken origin. Vet Microbiol 1997;54:195e200. Collier CT, van der Klis JD, Deplancke B, Anderson DB, Gaskins HR. Effects of tylosin on bacterial mucolysis, Clostridium perfringens colonization, and intestinal barrier function in a chick model of necrotic enteritis. Antimicrob Agents Chemother 2003;47:3311e7. Lovland A, Kaldhusdal M, Reitan LJ. Diagnosing Clostridium perfringens-associated necrotic enteritis in broiler flocks by an immunoglobulin G anti-alphatoxin enzyme-linked immunosorbent assay. Avian Pathol 2003;32:527e34. Cooper KK, Songer JG. Necrotic enteritis in chickens: a paradigm of enteric infection by Clostridium perfringens type A. Anaerobe 2009;15:55e60. Fukata T, Hadate Y, Baba E, Uemura T, Arakawa A. Influence of Clostridium perfringens and its toxin in germ-free chickens. Res Vet Sci 1988;44:68e70. Van Immerseel F, Rood JI, Moore RJ, Titball RW. Rethinking our understanding of the pathogenesis of necrotic enteritis in chickens. Trends Microbiol 2009;17:32e6. Cooper KK, Trinh HT, Songer JG. Immunization with recombinant alpha toxin partially protects broiler chicks against experimental challenge with Clostridium perfringens. Vet Microbiol 2009;133:92e7. Zekarias B, Mo H, Curtiss 3rd R. Recombinant attenuated Salmonella enterica serovar typhimurium expressing the carboxy-terminal domain of alpha toxin from Clostridium perfringens induces protective responses against necrotic enteritis in chickens. Clin Vaccine Immunol 2008;15:805e16. Kulkarni RR, Parreira VR, Sharif S, Prescott JF. Immunization of broiler chickens against Clostridium perfringens-induced necrotic enteritis. Clin Vaccine Immunol 2007;14:1070e7. Thompson DR, Parreira VR, Kulkarni RR, Prescott JF. Live attenuated vaccine-based control of necrotic enteritis of broiler chickens. Vet Microbiol 2006;113:25e34. Keyburn AL, Sheedy SA, Ford ME, Williamson MM, Awad MM, Rood JI, et al. Alpha-toxin of Clostridium perfringens is not an essential virulence factor in necrotic enteritis in chickens. Infect Immun 2006;74:6496e500. Cooper KK, Trinh H, Songer JG. Experimental necrotic enteritis in poultry. ClostPath: 5th International Meeting on the Molecular Biology and Pathogenesis of the Clostridia. Nottingham, UK, 2006. Cooper KK, Keel MK, Stewart B, Jost BH, Trinh H, Songer JG. Virulence for chickens of Clostridium perfringens isolated from poultry and other sources. 85th Annual Meeting of CRWAD: Conference of Research Workers in Animal Disease. Chicago, IL, 2004. Keyburn AL, Boyce JD, Vaz P, Bannam TL, Ford ME, Parker D, et al. NetB, a new toxin that is associated with avian necrotic enteritis caused by Clostridium perfringens. PLoS Pathog 2008;4:e26. Meer RR, Songer JG. Multiplex polymerase chain reaction assay for genotyping Clostridium perfringens. Am J Vet Res 1997;58:702e5. Lin YT, Labbe R. Enterotoxigenicity and genetic relatedness of Clostridium perfringens isolates from retail foods in the United States. Appl Environ Microbiol 2003;69:1642e6. Justin N, Walker N, Bullifent HL, Songer G, Bueschel DM, Jost H, et al. The first strain of Clostridium perfringens isolated from an avian source has an alphatoxin with divergent structural and kinetic properties. Biochemistry 2002;41:6253e62. Shimizu T, Ohtani K, Hirakawa H, Ohshima K, Yamashita A, Shiba T, et al. Complete genome sequence of Clostridium perfringens, an anaerobic flesheater. Proc Natl Acad Sci U S A 2002;99:996e1001. Timbermont L, Lanckriet A, Gholamiandehkordi AR, Pasmans F, Martel A, Haesebrouck F, et al. Origin of Clostridium perfringens isolates determines the ability to induce necrotic enteritis in broilers. Comp Immunol Microbiol Infect Dis; 2008. Shane SM, Koetting DG, Harrington KS. The occurrence of Clostridium perfringens in the intestine of chicks. Avian Dis 1984;28:1120e4.
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Anaerobe journal homepage: www.elsevier.com/locate/anaerobe
Pathogenesis and Toxins
Virulence for chickens of Clostridium perfringens isolated from poultry and other sources Kerry K. Cooper, James R. Theoret, Bernard A. Stewart, Hien T. Trinh, Robert D. Glock, J. Glenn Songer* Department of Veterinary Science and Microbiology, University of Arizona, 1117 E. Lowell St., Tucson, AZ 85721, USA
a r t i c l e i n f o
a b s t r a c t
Article history: Received 20 September 2009 Received in revised form 17 February 2010 Accepted 21 February 2010 Available online 1 March 2010
Clostridium perfringens type A is the most common cause of poultry necrotic enteritis (NE). Of the four “major” toxins, type A strains produce only alpha toxin (CPA), which has long been considered a major factor in pathogenesis of NE. We investigated the virulence for poultry of type A strains from a variety of enteric sources. Newly-hatched Cornish Rock chicks were fed a low protein diet for one week, a high protein diet for a second week, and then challenged with log-phase cultures of C. perfringens, mixed 3:4 (v/v) with high protein feed. Strain JGS4143 [genotype A, beta2 positive (cpb2pos), from a field case of NE] produced gross lesions compatible with NE in >85% of challenged birds. However, strains JGS1714 (enterotoxigenic genotype A, cpb2pos, human food poisoning), JGS1936 (genotype A, cpb2neg, bovine neonatal enteritis), JGS4142 (genotype A, cpb2pos, bovine jejunal hemorrhage syndrome), JGS1473 (genotype A, cpb2pos, chicken normal flora), JGS1070 (genotype C, cpb2pos, porcine hemorrhagic enteritis), JGS1882 (genotype A, cpb2pos, porcine neonatal enteritis), JGS1120 (ATCC 13124, genotype A, cpb2neg, gas gangrene), JGS4151 (strain 13, genotype A, cpb2pos, canine), and JGS4303 (SM101, enterotoxigenic genotype A, cpb2neg, human food poisoning) failed to produce disease. In vivo passage failed to increase virulence of the non-NE strains. NE strains must have specific poultry-associated virulence attributes, such as the recently identified NetB and other factors, which allow for the development of disease. Ó 2010 Elsevier Ltd. All rights reserved.
Keywords: Clostridium perfringens Poultry Necrotic enteritis Broiler chickens Virulence
1. Introduction Poultry necrotic enteritis (NE) is most commonly caused by Clostridium perfringens type A [1e5], although occasional cases are due to type C infection [6e10]. This species is anaerobic, sporeforming, gram-positive, and rod-shaped, and is divided into five toxinogenic types based on the pattern of production of four major toxins [4,11]. Control of NE has been most commonly achieved by use of antimicrobials [3]. However, concerns over downstream human health effects, such as resistance transfer from animal pathogens to human pathogens, have led to European Union bans on use of antimicrobial growth promoters [12e15]. A further increase in incidence of NE has also resulted from the introduction of products for immunoprophylaxis of coccidiosis [16,17]. Attenuated coccidia in vaccines may produce transient damage to intestinal epithelia, facilitating establishment of C. perfringens and production of NE [5,15,18,19]. A more important factor may be cessation of the use of
* Corresponding author. Tel.: þ1 520 621 2962; fax: þ1 520 621 6366. E-mail address: [email protected] (J.G. Songer). 1075-9964/$ e see front matter Ó 2010 Elsevier Ltd. All rights reserved. doi:10.1016/j.anaerobe.2010.02.006
ionophore coccidiostats. These compounds have been extraordinarily effective in preventing losses due to infection by Eimeria spp, but have also provided serendipitous anti-clostridial effects [15,20e23]. Consequently, NE has re-emerged as a major problem for the poultry industry [5,12,20,24,25]. Alpha toxin (CPA) has long been considered a critical virulence factor in the pathogenesis of NE, although specific evidence has been lacking. CPA is the only “major” toxin produced by type A strains, and higher levels have been detected in birds with NE than in normal birds [4,26]. Oral challenge of germ-free chicks with crude toxin preparations has resulted in disease development, and these effects were neutralized by anti-CPA serum mixed with the crude toxin prior to challenge [3,27]. Additionally, birds recovering from NE have high levels of anti-CPA antibodies, whereas healthy birds have low to undetectable levels [26,28]. Several studies have found that vaccinating birds with CPA provides partial protection against NE [29e31]. The role of CPA has recently been called into question. Vaccination with a CPA-deficient mutant protected against experimental challenge [32]. Seemingly more conclusive evidence was provided by the finding that an engineered CPA mutant of a virulent NE strain produced disease [33].
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A vital element in the development of prevention and control measures for NE is a better understanding of pathogenesis. We developed an experimental model of C. perfringens type A infection; strains isolated from field cases of NE are virulent, although variably so (unpublished data). Earlier, we reported results of preliminary work, demonstrating that virulence of C. perfringens type A for chickens is strain specific [34,35] and formalize those findings here. The overall objective of this work was to determine if CPAproducing type A strains from a variety of sources are virulent for chickens in our experimental model. Type A strains from normal chickens or from enteric disease in other species do not produce NE in chicks. This adds evidence to the argument that the virulence armada of NE strains includes factors other than CPA, including in many cases NetB [36] and other factors (unpublished data). 2. Methods and materials 2.1. Birds and care Commercial Jumbo Cornish Rock broiler chicks were obtained as day-old hatchlings from Murray McMurray Hatchery (Webster City, IA, USA). Birds were housed in 1.5 m diameter brooders constructed of 3 mm thick pegboard and divided into three equal parts. Commercial wood shavings provided as bedding were changed at two week intervals. Birds were fed a commercial chick starter ration (20% protein, Eagle Milling, Casa Grande, AZ, USA) through experimental day 7. Thereafter, birds were fed a high protein feed (28% protein) mixed 50:50 with menhaden fishmeal (SeaLac, Omega Protein, Houston, TX, USA). On day 14, feed was withheld for 20 h, and birds were then challenged with C. perfringens. Water was available ab lib in galvanized steel automatic waterers throughout each study. 2.2. Challenge inoculum and protocol Strains used in these studies are listed in Table 1, with host and disease (if any) of origin. Strains were stored at 80 C in 50% glycerol:50% brain heart infusion (BHI; Difco, Detroit, MI, USA). For inoculum preparation, approximately 10 ml were streaked for isolation on a plate of BHI agar with 5% citrated bovine blood. After incubation under anaerobic conditions (5% H2: 5% CO2: 90% N2) at 37 C for 24 h, 1e2 colonies were transferred into 10 ml of cooked meat medium (CMM, Difco) in Hungate tubes and incubated in the same atmosphere at 37 C for 18 h. The resulting culture was used to inoculate 100 ml fluid thioglycollate broth (FTG, Difco), which, after incubation as before, was diluted 1:10 in CMM and incubated as before. Thirty-three millilitre of the CMM culture were used as inoculum for 1 l FTG medium, and after 18 h incubation, this was mixed with feed for challenge. A separate serially-passed culture was prepared for each challenge feeding (total n ¼ 8). Numbers of Table 1 Clostridium perfringens strains. Genotype
Source of strains
netB
JGS1070 JGS1120 JGS1473 JGS1714 JGS1882 JGS1936 JGS4303 JGS4142 JGS4143 JGS4151
C, cpb2þ A A, cpb2þ A, cpeþ A, cpb2þ A A, cpeþ A, cpb2þ A, cpb2þ A, cpb2þ
Porcine hemorrhagic enteritis ATCC 13124a Avian normal flora Human foodborne disease Porcine neonatal enteritis Bovine neonatal enteritis SM101a Bovine jejunal hemorrhagic syndrome Field case NE Strain 13b
Neg. Neg. Neg. Neg. Neg. Neg. Neg. Neg. Pos. Neg.
a
Sequenced (Ref. [35]). Sequenced (Ref. [41]).
2.3. PCR-C. perfringens genotyping [37] Isolated colonies on BHI agar with 5% citrated bovine blood were suspended in 200 ml of HPLC-grade water (Mallinckrodt, Hazelwood, MO, USA), boiled for 20 min, and centrifuged 13,000 g for 2 min. The supernatant fluid served as template DNA, and 5 ml were added to 20 ml of a master mix solution containing primers, Taq polymerase (Promega, Madison, WI, USA), MgCl2 (25 mM, Sigma-Aldrich), dNTPs (5 mM, Promega), and 10 Assay Buffer (Promega), to give a total reaction volume of 25 ml. PCR amplification was comprised of 35 cycles of denaturing (94 C, 1 min), primer annealing (55 C, 1 min) and extension (72 C, 1 min). Aliquots (15 ml) of each PCR product were loaded into wells of a 1.5% (w/v) agarose gel (GenePure LE, ISC BioExpress, Kaysville, UT, USA), stained with 0.01% (v/v) ethidium bromide (Sigma-Aldrich), and separated by electrophoresis. PCR products were visualized via UV transillumination and digitally photographed. 2.4. Pulsed-field gel electrophoresis (PFGE) Genotyped strains were embedded in plugs of 2% CleanCut agarose (Bio-Rad, Hercules, CA, USA), according to the method of Lin and Labbe [38]. After cell lysis and DNA digestion with SmaI (New England Biolabs, Ipswich, MA, USA), electrophoresis was performed on a CHEF Mapper DRII (Bio-Rad) at 6.0 V/cm for 20 h with an angle of 120 , an initial pulse time of 0.50 s, and a final pulse time of 40 s. After staining with ethidium bromide, gels were examined and photographed via UV transillumination. 2.5. Assays of alpha toxin (CPA; lecithinase) activity [39]
JGS #
b
colony-forming units (cfu) were determined by plating serial 10fold dilutions on BHI agar. Birds were inoculated on days 15e18. High protein feed and FTG medium culture were mixed in a ratio of 3:4 (v/v). The mixture, which had a paste-like consistency, was then placed in galvanized steel feed trays and offered to chicks. Trays were cleaned and remaining feed disposed of prior to each subsequent feeding. Negative control birds were challenged with un-inoculated FTG mixed with high protein feed at the same ratio. On day 19, birds were euthanized by CO2 asphyxiation. Necrotic intestinal lesions were scored (0: no gross lesions; 1þ: thin-walled or friable small intestine; 2þ: focal necrosis or ulceration; 3þ: large patches of necrosis; 4þ: severe or extensive necrosis typical of field cases) and segments fixed in 10% buffered formalin or retained fresh for bacteriological culture. Intestinal contents serially diluted in phosphate buffered saline [PBS; 0.01 M, pH 7.2] were plated on C. perfringens selective agar (CPSA [BHI agar supplemented with 0.5% yeast extract (Becton Dickinson, Franklin Lakes, NJ, USA), 0.1% sodium metabisulfite (Sigma-Aldrich, St. Louis, MO, USA), 5% citrated bovine blood, and D-cycloserine (400 mg/ml; Sigma-Aldrich)] and incubated under anaerobic conditions (as above) for 24 h.
Egg yolk emulsion was prepared by adding one asepticallyremoved egg yolk to 20 ml DMG buffer [0.04 M 3,3 dimethylglutaric acid (Sigma-Aldrich) pH 7.0 plus 0.008 M CaCl2 (Sigma-Aldrich), 0.0002 M ZnSO4 (Sigma-Aldrich), and 0.1% bovine serum albumin (Sigma-Aldrich); pH adjusted to 7.2 and volume adjusted to 100 ml with HPLC-grade water] and mixed thoroughly and centrifuged (26,890 g, 20 C, 20 min). Supernatant was diluted 1:10 in DMG buffer prior to use. Two-fold serial dilutions (1:2e1:256) of purified recombinant CPA or test samples were prepared in DMG buffer, and 100 ml of each were added to wells of a 96-well plate. An aliquot (100 ml) of egg yolk emulsion was then
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added to each well and the plate sealed to prevent evaporation. After incubation at 37 C for 1 h, results were read spectrophotometrically at 450 nm. Amounts of CPA in test samples were derived by comparison of results to a plot generated by testing purified recombinant CPA standards. 2.6. Institutional animal care and use committee (IACUC) approval Studies presented here were pre-reviewed and pre-approved by the University of Arizona IACUC, under protocol number 02-204. 3. Results We examined the ability of isolates of C. perfringens (Table 1) to produce NE in our model. Disease did not result from challenge with these isolates, although 58/66 (87.9%) positive controls (inoculated with NE isolate JGS4143) developed gross lesions (Fig. 1) with an average lesion score of 2.31 (Table 2, Fig. 2). Negative controls (challenged with un-inoculated thioglycollate broth) did not develop lesions. There were no statistically significant differences in CPA production across strains and, in fact, positive control strain JGS4143 produced less CPA than any other strain in vitro (Table 2). Intestinal samples from challenged birds were examined by bacteriologic culture for C. perfringens, and PCR-genotyped and recovered isolates were confirmed as the challenge strain in each case by comparing PFGE profiles. Attempts to recover challenge strains in initial trials were successful only from birds inoculated with strains JGS4143 and JGS1473 (avian normal flora). Further groups of birds were challenged with JGS1070, JGS1714, JGS1882, JGS1936, and JGS4142, as well as strain JGS4151 [strain 13 [40]]. No challenged birds developed lesions, but PFGE analysis of at least one-half of the isolates (up to 100 per bird) showed that we recovered all challenge isolates except JGS1070 (a type C porcine isolate). Only avian isolates (JGS4143 and JGS1473) were isolated from the jejunum, while JGS1714 was only recovered from the duodenum of challenged birds. The other non-avian isolates (JGS1882, JGS1936, JGS4142, JGS4151) were isolated from the ileum of challenged birds. Birds were then inoculated with these isolates (JGS1882, JGS1714, JGS1936, JGS4142, and JGS4151) and JGS1473, all after passage through chicks, to determine if in vivo passage led to detectable virulence. Again, no challenged birds developed lesions. Positive control birds challenged with JGS4143 developed gross lesions [15/16 (93.7%), average lesion score 3.07] and negative controls did not.
Fig. 1. Macroscopic examination of the intestine in birds challenged with C. perfringens. A. Gross lesions typical of necrotic enteritis from bird challenged with JGS4143. B. Lack of gross lesions in intestine of bird challenged with JGS4142; this is typical of intestines of birds challenged with non-NE strains.
291
Table 2 Response of birds to challenge with non-avian strains of Clostridium perfringens. Strain
Birds with gross lesion/total (%)
Average lesion score SD
Average challenge inocula titer (log[10]) SD
Average challenge inocula CPA titer (mg/ml) SD
JGS1473 JGS1070 JGS1882 JGS4142 JGS1714 JGS1936 SM101 Strain 13 ATCC 13124 JGS4143 Negative control
0/16 (0.0%) 0/16 (0.0%) 0/16 (0.0%) 0/15 (0.0%) 0/16 (0.0%) 0/16 (0.0%) 0/18 (0.0%) 0/20 (0.0%) 0/18 (0.0%) 58/66 (87.9%) 0/67 (0.0%)
0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 2.31 0.50 0.0
6.98 1.6 5.70 0.41 5.86 0.99 5.88 0.88 5.88 0.87 8.60 0.47 N/A N/A N/A 8.48 0.65 N/A
884.33 42.52 903.63 83.69 921.58 40.2 923.25 74.57 943.33 46.25 932.92 59.14 N/A N/A N/A 844.42 33.27 N/A
4. Discussion The results of these studies confirm our earlier findings [34,35] and those of others [41] that not all type A strains of C. perfringens produce NE in birds under the challenge conditions used. In fact, we have shown that in vivo passage of NE strains can dramatically increase their virulence (unpublished), but in these studies, a single in vivo passage apparently did not alter virulence. All avirulent
Fig. 2. Microscopic examination of the intestine in birds challenged with C. perfringens. A. Intestine of bird challenged with JGS4143. Extensive fibrinonecrotic inflammation, with bacteria and distended crypts and inflammatory infiltrate in the lamina propria (40). B. Intestine of bird challenged with JGS1473. No microscopic lesions are observed. Findings typical of intestines from birds challenged with other non-NE strains (40).
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strains produced CPA, and none produced NetB [36], suggesting that CPA is not the primary virulence attribute. In addition, these results and the finding that inoculation of birds with CPAcontaining culture supernatant fluid did not produce gross lesions (unpublished data), led us to conclude that the quantity of CPA in experimental inocula does not significantly affect the ability of an isolate to produce disease. Only JGS4143 and JGS1473 were recovered from jejunum, the most common site of lesion development. JGS1882, JGS1936, JGS4142, and JGS4151 were isolated from the ileum post-challenge, which is in keeping with findings of others that C. perfringens most commonly colonizes the ileum and the ceca of chickens as part of the normal flora [42]. JGS1714 was isolated from the duodenum, but not the jejunum or the ileum, suggesting possible lack of colonization factors that might allow avian strains to colonize the jejunum. Additionally, the failure of non-avian strains to effectively colonize birds following challenge with high levels of inoculum also suggest an overall deficiency in poultry colonization factors. In conclusion, NE strains must have specific poultry-associated virulence attributes, such as the recently identified NetB and other factors, which allow for the development of disease. Additionally, failure of sequenced strains (Table 2) to produce disease suggests that they will provide an excellent background for comparisons with genome sequences of NE strains, possibly allowing identification of additional specific virulence factors. Acknowledgements
[15]
[16] [17]
[18] [19]
[20]
[21]
[22]
[23]
[24]
[25]
[26] [27]
The authors thank Dr. Sharon Dial for assistance with the histology from the studies, and Drs. Lynn Joens and Michael Riggs for helpful commentary. This research was funded in part by a grant from Schering-Plough Animal Health. References [1] Al-Sheikhly F, Truscott RB. The interaction of Clostridium perfringens and its toxins in the production of necrotic enteritis of chickens. Avian Dis 1977;21:256e63. [2] Lovland A, Kaldhusdal M. Liver lesions seen at slaughter as an indicator of necrotic enteritis in broiler flocks. FEMS Immunol Med Microbiol 1999;24:345e51. [3] Lovland A, Kaldhusdal M, Redhead K, Skjerve E, Lillehaug A. Maternal vaccination against subclinical necrotic enteritis in broilers. Avian Pathol 2004;33:83e92. [4] Songer JG. Clostridial enteric diseases of domestic animals. Clin Microbiol Rev 1996;9:216e34. [5] Williams RB, Marshall RN, La Ragione RM, Catchpole J. A new method for the experimental production of necrotic enteritis and its use for studies on the relationships between necrotic enteritis, coccidiosis and anti-coccidial vaccination of chickens. Parasitol Res 2003;90:19e26. [6] Gardiner MR. Clostridial infections in poultry in Western Australia. Aust Vet J 1967;43:359e60. [7] Kaldhusdal M, Hofshagen M, Lovland A, Langstrand H, Redhead K. Necrotic enteritis challenge models with broiler chickens raised on litter: evaluation of preconditions, Clostridium perfringens strains and outcome variables. FEMS Immunol Med Microbiol 1999;24:337e43. [8] Kaldhusdal M, Schneitz C, Hofshagen M, Skjerve E. Reduced incidence of Clostridium perfringens-associated lesions and improved performance in broiler chickens treated with normal intestinal bacteria from adult fowl. Avian Dis 2001;45:149e56. [9] Kwatra MS, Chaudhury B. A presumptive diagnosis in the fowl (Gallus gallus domesticus) from Assam (India). Avian Dis 1976;20:401e6. [10] Prescott JF, Sivendra R, Barnum DA. The use of bacitracin in the prevention and treatment of experimentally-induced necrotic enteritis in the chicken. Can Vet J 1978;19:181e3. [11] Nauerby B, Pedersen K, Madsen M. Analysis by pulsed-field gel electrophoresis of the genetic diversity among Clostridium perfringens isolates from chickens. Vet Microbiol 2003;94:257e66. [12] Casewell M, Friis C, Marco E, McMullin P, Phillips I. The European ban on growth-promoting antibiotics and emerging consequences for human and animal health. J Antimicrob Chemother 2003;52:159e61. [13] Emborg H, Ersboll AK, Heuer OE, Wegener HC. The effect of discontinuing the use of antimicrobial growth promoters on the productivity in the Danish broiler production. Prev Vet Med 2001;50:53e70. [14] Emborg HD, Andersen JS, Seyfarth AM, Wegener HC. Relations between the consumption of antimicrobial growth promoters and the occurrence of
[28] [29]
[30]
[31]
[32]
[33]
[34]
[35]
[36]
[37] [38]
[39]
[40]
[41]
[42]
resistance among Enterococcus faecium isolated from broilers. Epidemiol Infect 2004;132:95e105. Van Immerseel F, De Buck J, Pasmans F, Huyghebaert G, Haesebrouck F, Ducatelle R. Clostridium perfringens in poultry: an emerging threat for animal and public health. Avian Pathol 2004;33:537e49. Weber FH, Evans NA. Immunization of broiler chicks by in ovo injection of Eimeria tenella sporozoites, sporocysts, or oocysts. Poult Sci 2003;82:1701e7. Weber FH, Genteman KC, LeMay MA, Lewis Sr DO, Evans NA. Immunization of broiler chicks by in ovo injection of infective stages of Eimeria. Poult Sci 2004;83:392e9. Williams RB. Anticoccidial vaccines for broiler chickens: pathways to success. Avian Pathol 2002;31:317e53. Martel A, Devriese LA, Cauwerts K, De Gussem K, Decostere A, Haesebrouck F. Susceptibility of Clostridium perfringens strains from broiler chickens to antibiotics and anticoccidials. Avian Pathol 2004;33:3e7. Grave K, Kaldhusdal MC, Kruse H, Harr LM, Flatlandsmo K. What has happened in Norway after the ban of avoparcin? Consumption of antimicrobials by poultry. Prev Vet Med 2004;62:59e72. Brennan J, Bagg R, Barnum D, Wilson J, Dick P. Efficacy of narasin in the prevention of necrotic enteritis in broiler chickens. Avian Dis 2001;45: 210e4. Elwinger K, Schneitz C, Berndtson E, Fossum O, Teglof B, Engstom B. Factors affecting the incidence of necrotic enteritis, caecal carriage of Clostridium perfringens and bird performance in broiler chicks. Acta Vet Scand 1992;33:369e78. Watkins KL, Shryock TR, Dearth RN, Saif YM. In-vitro antimicrobial susceptibility of Clostridium perfringens from commercial turkey and broiler chicken origin. Vet Microbiol 1997;54:195e200. Collier CT, van der Klis JD, Deplancke B, Anderson DB, Gaskins HR. Effects of tylosin on bacterial mucolysis, Clostridium perfringens colonization, and intestinal barrier function in a chick model of necrotic enteritis. Antimicrob Agents Chemother 2003;47:3311e7. Lovland A, Kaldhusdal M, Reitan LJ. Diagnosing Clostridium perfringens-associated necrotic enteritis in broiler flocks by an immunoglobulin G anti-alphatoxin enzyme-linked immunosorbent assay. Avian Pathol 2003;32:527e34. Cooper KK, Songer JG. Necrotic enteritis in chickens: a paradigm of enteric infection by Clostridium perfringens type A. Anaerobe 2009;15:55e60. Fukata T, Hadate Y, Baba E, Uemura T, Arakawa A. Influence of Clostridium perfringens and its toxin in germ-free chickens. Res Vet Sci 1988;44:68e70. Van Immerseel F, Rood JI, Moore RJ, Titball RW. Rethinking our understanding of the pathogenesis of necrotic enteritis in chickens. Trends Microbiol 2009;17:32e6. Cooper KK, Trinh HT, Songer JG. Immunization with recombinant alpha toxin partially protects broiler chicks against experimental challenge with Clostridium perfringens. Vet Microbiol 2009;133:92e7. Zekarias B, Mo H, Curtiss 3rd R. Recombinant attenuated Salmonella enterica serovar typhimurium expressing the carboxy-terminal domain of alpha toxin from Clostridium perfringens induces protective responses against necrotic enteritis in chickens. Clin Vaccine Immunol 2008;15:805e16. Kulkarni RR, Parreira VR, Sharif S, Prescott JF. Immunization of broiler chickens against Clostridium perfringens-induced necrotic enteritis. Clin Vaccine Immunol 2007;14:1070e7. Thompson DR, Parreira VR, Kulkarni RR, Prescott JF. Live attenuated vaccine-based control of necrotic enteritis of broiler chickens. Vet Microbiol 2006;113:25e34. Keyburn AL, Sheedy SA, Ford ME, Williamson MM, Awad MM, Rood JI, et al. Alpha-toxin of Clostridium perfringens is not an essential virulence factor in necrotic enteritis in chickens. Infect Immun 2006;74:6496e500. Cooper KK, Trinh H, Songer JG. Experimental necrotic enteritis in poultry. ClostPath: 5th International Meeting on the Molecular Biology and Pathogenesis of the Clostridia. Nottingham, UK, 2006. Cooper KK, Keel MK, Stewart B, Jost BH, Trinh H, Songer JG. Virulence for chickens of Clostridium perfringens isolated from poultry and other sources. 85th Annual Meeting of CRWAD: Conference of Research Workers in Animal Disease. Chicago, IL, 2004. Keyburn AL, Boyce JD, Vaz P, Bannam TL, Ford ME, Parker D, et al. NetB, a new toxin that is associated with avian necrotic enteritis caused by Clostridium perfringens. PLoS Pathog 2008;4:e26. Meer RR, Songer JG. Multiplex polymerase chain reaction assay for genotyping Clostridium perfringens. Am J Vet Res 1997;58:702e5. Lin YT, Labbe R. Enterotoxigenicity and genetic relatedness of Clostridium perfringens isolates from retail foods in the United States. Appl Environ Microbiol 2003;69:1642e6. Justin N, Walker N, Bullifent HL, Songer G, Bueschel DM, Jost H, et al. The first strain of Clostridium perfringens isolated from an avian source has an alphatoxin with divergent structural and kinetic properties. Biochemistry 2002;41:6253e62. Shimizu T, Ohtani K, Hirakawa H, Ohshima K, Yamashita A, Shiba T, et al. Complete genome sequence of Clostridium perfringens, an anaerobic flesheater. Proc Natl Acad Sci U S A 2002;99:996e1001. Timbermont L, Lanckriet A, Gholamiandehkordi AR, Pasmans F, Martel A, Haesebrouck F, et al. Origin of Clostridium perfringens isolates determines the ability to induce necrotic enteritis in broilers. Comp Immunol Microbiol Infect Dis; 2008. Shane SM, Koetting DG, Harrington KS. The occurrence of Clostridium perfringens in the intestine of chicks. Avian Dis 1984;28:1120e4.