Virulence of Clostridium perfringens in an experimental model of poultry necrotic enteritis

Department of Veterinary Science and Microbiology, University of Arizona, Tucson, AZ 85721, USA.
Veterinary Microbiology (Impact Factor: 2.51). 10/2009; 142(3-4):323-8. DOI: 10.1016/j.vetmic.2009.09.065
Source: PubMed


Poultry necrotic enteritis (NE) has, over recent decades, been prevented and treated by addition of antimicrobials to poultry feed. Recent bans of antimicrobial growth promoters in feed, as well as other factors, have led to a slow, worldwide re-emergence of NE. Understanding of pathogenesis of NE has been hampered by lack of a consistent and effective experimental model in which virulence of strains can be reliably evaluated, with an endpoint yielding lesions comparable to those seen in acute NE in the field. The overall objective of this work was to develop an experimental approach that would allow consistent production of a full range of clinical signs and lesions of the disease, and to do so without use of coccidia as inciting agents. In addition, we assessed the virulence of strains of Clostridium perfringens from field cases of NE. Broiler chicks fed a commercial chick starter for 7 days post-hatch were switched to a high protein feed mixed 50:50 with fishmeal for an additional 7 days. On day 14, feed was withheld for 20 h, and birds were then offered feed mixed with C. perfringens (3 parts culture to 4 parts feed) twice daily on 4 consecutive days. On average, >75% of challenged birds developed typical gross lesions when inoculated with type A strains from field cases of NE. In addition, in vivo passage apparently increases strain virulence. Virulence varies from strain-to-strain; NetB-producing strains were virulent, as were some NetB non-producing strains.

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    • "Importantly, similar microscopic lesions observed in the duodenum are consistent with lesions seen in field cases of necrotic enteritis and in experimental infections following inoculation with C. perfringens bacteria (Truscott and Al-Sheikhly, 1977; Olkowski et al., 2008). Exposure to C. perfringens culture supernatant here did not appear to induce local heterophil recruitment in the duodenum; an increase in heterophil numbers has been observed in the blood of broilers 3 d after C. perfringens bacterial challenge (Cooper and Songer, 2010), but clearly the absence of bacterial cells did not instigate their localized recruitment here. Heterophils are the main polymorphonuclear leukocytes in poultry and act as the first line of defense against microorganisms (Montali, 1988). "
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    ABSTRACT: type A is the main etiological factor for necrotic enteritis, a multifactorial enteric disease that penalizes performance, health, and welfare of poultry. Lack of knowledge of host responses and disease pathogenesis is slowing down progress on developing therapies for disease control. A combined genomewide and targeted gene approach was used to investigate pathways and biological functions affected by the infusion of culture supernatant in the duodenum of broilers in 2 experiments. An in situ isolated loop of duodenum was prepared in anesthetized broilers of 3 wk of age (Exp. 1) and was infused either with crude culture supernatant ( = 7; treated), positive for necrotic enteritis B-like toxin (NetB) as determined by a cytotoxicity assay, or with a control preparation ( = 6; control). Birds were maintained alive for 1 h and then euthanized for tissue recovery. The use of the Affymetrix chicken genome array on RNA samples from loop tissue showed top biological functions affected by culture supernatant infusion included cell morphology, immune cell trafficking, and cell death; pathways affected included death receptor signaling, inflammatory response, and nuclear factor (NF)-κB signaling. In a second in situ study (Exp. 2), broilers were maintained alive for 4 h to monitor temporal expression patterns of targeted genes. Duodenal tissue was removed at 0.5, 1, 2, and 4 h post-infusion with culture supernatant ( = 9) or a control preparation ( = 5) for histology and gene expression analysis. Genes encoding proinflammatory cytokines, such as interferon γ (γ), cell trafficking, such as neuroblastoma 1 () and B cell CLL/Lymphoma 6 (), and cell death, such as Fas cell surface death receptor () and GTPase IMAP family member 8 (), were differentially expressed in the duodenum of treated and control broilers ( < 0.05). We have demonstrated that culture supernatant (NetB positive) infusion resulted in histological and gene expression changes consistent with necrotic enteritis in the duodenum of broilers. In the absence of live bacteria, crude culture supernatant resulted in early immunomodulation, inflammation, and cell death in the duodenum. The pathways identified here can be targeted for the development of new drugs, vaccines, and novel therapies for necrotic enteritis in broilers.
    Full-text · Article · Jun 2015 · Journal of Animal Science
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    • "Two different challenge methods were used: a direct oral gavage challenge with small volumes of culture, which may be more akin to challenges that occur in the field, and a large volume in-feed challenge that gives a more robust challenge, resulting in higher levels of disease in a greater percentage of birds. These oral gavage and in-feed necrotic enteritis disease induction models were performed essentially as previously described [10,20], with some modification to the timing to fit in with vaccination schedules. Briefly, groups of 10 chickens were kept in adjacent, but separate, pens in an animal isolation facility. "
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    ABSTRACT: NetB toxin from Clostridium perfringens is a major virulence factor in necrotic enteritis in poultry. In this study the efficacy of NetB as a vaccine antigen to protect chickens from necrotic enteritis was examined. Broiler chickens were immunized subcutaneously with purified recombinant NetB (rNetB), formalin treated bacterin and cell free toxoid with or without rNetB supplementation. Intestinal lesion scores and NetB antibody levels were measured to determine protection after mild oral gavage, moderate in-feed and heavy in-feed challenges with virulent C. perfringens isolates. Birds immunized with rNetB were significantly protected against necrotic enteritis when challenged with a mild oral dose of virulent bacteria, but were not protected when a more robust challenge was used. Bacterin and cell free toxoid without rNetB supplementation did not protect birds from moderate and severe in-feed challenge. Only birds immunized with bacterin and cell free toxoid supplemented with rNetB showed significant protection against moderate and severe in-feed challenge, with the later giving the greatest protection. Higher NetB antibody titres were observed in birds immunized with rNetB compared to those vaccinated with bacterin or toxoid, suggesting that the in vitro levels of NetB produced by virulent C. perfringens isolates are too low to induce the development of a strong immune response. These results suggest that vaccination with NetB alone may not be sufficient to protect birds from necrotic enteritis in the field, but that in combination with other cellular or cell-free antigens it can significantly protect chickens from disease.
    Full-text · Article · Jul 2013 · Veterinary Research
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    • "The role of the netB toxin in NE in turkeys needs to be further investigated. Key words: Clostridium perfringens , turkey , necrotic enteritis Truscott, 1977), but Keyburn et al. (2006) and Cooper et al. (2010) showed that α-toxin may not be an essential causative factor of NE. More recently, a novel toxin, NE toxin B (NetB), has been discovered and strongly associated with the pathogenesis of NE. "
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    ABSTRACT: Clostridium perfringens is an important bacterial pathogen, especially in poultry, where it can lead to both subclinical and clinical disease. The aim of this study was to present data on pathological findings at outbreaks of necrotic enteritis (NE) in turkey production in Finland during the period from 1998 to 2012. Furthermore, C. perfringens isolates from healthy and diseased turkeys were characterized and their genetic diversity was investigated using pulsed-field gel electrophoresis (PFGE). Isolates (n = 212) from birds with necrotic gut lesions and from healthy flocks of 30 commercial turkey farms were characterized for the presence of cpa, cpb, iA, etx, cpb2, and cpe and netB genes. A total of 93 C. perfringens isolates, including 55 from birds with necrotic gut lesions and 38 from healthy birds from 13 different farms, were analyzed with PFGE. All contract turkey farmers (n = 48) of a turkey company that produces 99% of domestic turkey meat in Finland were interviewed about background information, management at the farm, and stress factors related to NE outbreaks. Pulsed-field gel electrophoresis analysis with SmaI restriction enzyme resulted in 30 PFGE patterns among the 92 C. perfringens isolates of high diversity. Out of all isolates, 212 (100%) were α-toxin-positive and one isolate (0.5%) was both α- and β2 toxin-positive. Fourteen isolates (6.6%) were necrotic enteritis toxin B (NetB) positive; all were recovered from turkeys with NE. In none of the isolates obtained from healthy turkeys was the netB toxin identified. In conclusion, a high diversity of C. perfringens isolates from turkeys with different health status was shown. All isolates produced α toxin, whereas only low percentages of isolates carried the netB toxin gene. The role of the netB toxin in NE in turkeys needs to be further investigated.
    Preview · Article · Jul 2013 · Poultry Science
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