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      Developing an experimental necrotic enteritis model in turkeys - the impact of Clostridium perfringens, Eimeria meleagrimitis and host age on frequency of severe intestinal lesions

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          Abstract

          Background

          Necrotic enteritis is a significant problem to the poultry industry globally and, in Norway up to 30% of Norwegian turkey grow-outs can be affected. However, despite an awareness that differences exist between necrotic enteritis in chickens and turkeys, little information exists concerning the pathogenesis, immunity, microbiota or experimental reproduction of necrotic enteritis in turkeys. In particular, it is important to determine the appearance of the gross lesions, the age dependency of the disease and the role of netB toxin of Clostridium perfringens. To this end, we report our findings in developing an in vivo experimental model of necrotic enteritis in turkeys.

          Results

          A four tier (0–3) scoring system with clearly defined degrees of severity of macroscopic intestinal lesions was developed, based on 2312 photographic images of opened intestines from 810 B.U.T. 10 or B.U.T. Premium turkeys examined in nine experiments. Loss of macroscopically recognizable villi in the anterior small intestine was established as the defining lesion qualifying for a score 3 (severe intestinal lesions). The developed scoring system was used to identify important factors in promoting high frequencies of turkeys with severe lesions: a combined Eimeria meleagrimitis and Clostridium perfringens challenge, challenge at five rather than 3 weeks of age, the use of an Eimeria meleagrimitis dose level of at least 5000 oocysts per bird and finally, examination of the intestines of 5-week-old turkeys at 125 to 145 h after Eimeria meleagrimitis inoculation. Numbers of oocysts excreted were not influenced by Clostridium perfringens inoculation or turkey age. Among three different lesion score outcomes tested, frequency of severe lesions proved superior in discriminating between impact of four combinations of Clostridium perfringens inoculation and turkey age at challenge.

          Conclusions

          This study provides details for the successful establishment of an in vivo model of necrotic enteritis in turkeys.

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          Most cited references16

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          The successful experimental induction of necrotic enteritis in chickens by Clostridium perfringens: a critical review

          Necrotic enteritis (NE) is one of the most important enteric diseases in poultry and is a high cost to the industry worldwide. It is caused by avian-specific, Necrotic Enteritis Beta toxin (NetB)-producing, strains of Clostridium perfringens that also possess in common other virulence-associated genes. In Europe the disease incidence has increased since the ban on in-feed “growth promoting” antibiotics. Because of this, many recent studies of NE have focused on finding different ways to control the disease, and on understanding its pathogenesis. Frustratingly, reproduction of the disease has proven impossible for some researchers. This review describes and discusses factors known to be important in reproducing the disease experimentally, as well as other considerations in reproducing the disease. The critical bacterial factor is the use of virulent, netB-positive, strains; virulence can be enhanced by using tpeL- positive strains and by the use of young rather than old broth cultures to increase toxin expression. Intestinal damaging factors, notably the use of concurrent or preceding coccidial infection, or administration of coccidial vaccines, combined with netB-positive C. perfringens administration, can also be used to induce NE. Nutritional factors, particularly feeding high percentage of cereals containing non-starch polysaccharides (NSP) (wheat, rye, and barley) enhance disease by increasing digesta viscosity, mucus production and bacterial growth. Animal proteins, especially fish meal, enhance C. perfringens proliferation and toxin production. Other factors are discussed that may affect outcome but for which evidence of their importance is lacking. The review compares the different challenge approaches; depending on the aim of particular studies, the different critical factors can be adjusted to affect the severity of the lesions induced. A standardized scoring system is proposed for international adoption based on gross rather than histopathological lesions; if universally adopted this will allow better comparison between studies done by different researchers. Also a scoring system is provided to assist decisions on humane euthanasia of sick birds.
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            Two necrotic enteritis predisposing factors, dietary fishmeal and Eimeria infection, induce large changes in the caecal microbiota of broiler chickens.

            It is widely established that a high-protein fishmeal supplemented starter diet and Eimeria infection can predispose birds to the development of clinical necrotic enteritis symptoms following Clostridium perfringens infection. However, it has not been clearly established what changes these treatments cause to predispose birds to succumb to necrotic enteritis. We analysed caecal microbiota of 4 groups of broilers (n=12) using deep pyrosequencing of 16S rDNA amplicons: (1) control chicks fed a control diet, (2) Eimeria infected chicks fed control diet, (3) chicks fed fishmeal supplemented diet and lastly (4) both fishmeal fed and Eimeria infected chicks. We found that the high-protein fishmeal diet had a strong effect on the intestinal microbiota similar to the previously reported effect of C. perfringens infection. We noted major changes in the prevalence of various lactobacilli while the total culturable Lactobacillus counts remained stable. The Ruminococcaceae, Lachnospiraceae, unknown Clostridiales and Lactobacillaceae families were most affected by fishmeal with increases in a number of operational taxonomic units (OTUs) that had previously been linked to Crohn's disease and reductions in OTUs known to be butyrate producers. Eimeria induced very different changes in microbiota; Ruminococcaceae groups were reduced in number and three unknown Clostridium species were increased in abundance. Additionally, Eimeria did not significantly influence changes in pH, formic, propionic or isobutyric acid while fishmeal induced dramatic changes in all these measures. Both fishmeal feeding and Eimeria infection induced significant changes in the gut microbiota; these changes may play an important role in predisposing birds to necrotic enteritis. Copyright © 2014 Elsevier B.V. All rights reserved.
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              Microbial shifts associated with necrotic enteritis.

              An outbreak of necrotic enteritis (NE) is a complex process requiring one or a number of predisposing factors rather than just the presence of pathogenic Clostridium perfringens. Examples are dietary influences, such as high levels of non-starch polysaccharides and fishmeal, and factors that evoke epithelial cell damage, such as Fusarium mycotoxins in feed and Eimeria infections. Recent studies have shown that different predisposing factors induce similar shifts in the intestinal microbiota composition. Butyrate-producing-strains of the Ruminococcaceae family are decreased in abundance by both fishmeal and Eimeria. Similarly, a decreased abundance of butyrate-producing-strains belonging to the Lachnospiraceae family has been induced by fishmeal. Also shifts are observed in the lactic acid-producing bacteria, such as decreased abundance of Lactobacillus johnsonii or Weissella confusa, when broilers were fed a fishmeal-based diet or a Fusarium mycotoxin contaminated diet. Finally, the abundance of Candidatus Savagella was decreased in broilers following Eimeria challenge or feeding a fumonisins contaminated diet. The nature of the microbiota shifts indicate that immune modulatory actions of the intestinal microbiota may play a critical role in the effect on the necrosis inducing activity of C. perfringens. Indeed, colonization with butyrate-producing bacteria plays a key role in counteracting inflammation in the gut and preserving intestinal integrity, while Candidatus Savagella is involved in stimulating Th17 and immunoglobulin A responses. Lactic acid bacteria stimulate colonization of lactate-utilizing and butyrate-producing Lachnospiraceae. Future research needs to clarify the role of the microbiota changes in the pathogenesis of NE.
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                Author and article information

                Contributors
                S.P.Hardy@brighton.ac.uk
                sylvie.benestad@vetinst.no
                inger.hamnes@vetinst.no
                torfinn.moldal@vetinst.no
                robert.bruce.david@nortura.no
                jbarta@uoguelph.ca
                Jean-Michel.REPERANT@anses.fr
                magne.kaldhusdal@vetinst.no
                Journal
                BMC Vet Res
                BMC Vet. Res
                BMC Veterinary Research
                BioMed Central (London )
                1746-6148
                18 February 2020
                18 February 2020
                2020
                : 16
                : 63
                Affiliations
                [1 ]GRID grid.12477.37, ISNI 0000000121073784, University of Brighton, ; Lewes Road, Brighton, BN2 4GJ UK
                [2 ]GRID grid.410549.d, ISNI 0000 0000 9542 2193, Norwegian Veterinary Institute, ; P.O.B. 750 Sentrum, 0106 Oslo, Norway
                [3 ]GRID grid.457991.7, ISNI 0000 0000 8608 5359, Nortura SA, ; Sloraveien 60, 1878 Haerland, Norway
                [4 ]GRID grid.34429.38, ISNI 0000 0004 1936 8198, University of Guelph, ; 50 Stone Road East, Guelph, ON N1G 2W1 Canada
                [5 ]GRID grid.15540.35, ISNI 0000 0001 0584 7022, Anses, Laboratory of Ploufragan-Plouzané-Niort, ; 22440 Ploufragan, France
                Author information
                http://orcid.org/0000-0002-0498-1470
                Article
                2270
                10.1186/s12917-020-2270-5
                7029515
                32070340
                1721ea3d-9663-4ce9-8d51-cbf105b04668
                © The Author(s). 2020

                Open AccessThis article is distributed under the terms of the Creative Commons Attribution 4.0 International License ( http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver ( http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.

                History
                : 28 June 2019
                : 31 January 2020
                Funding
                Funded by: FundRef http://dx.doi.org/10.13039/501100005416, Norges Forskningsråd;
                Award ID: 225177
                Award Recipient :
                Funded by: Norwegian Agricultural and Food Research Fund
                Award ID: 225177
                Award Recipient :
                Categories
                Research Article
                Custom metadata
                © The Author(s) 2020

                Veterinary medicine
                necrotic enteritis,challenge model,turkeys,macroscopic lesions,scoring system,clostridium perfringens,eimeria meleagrimitis,age,refinement

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