麻醉特异性病原体游离鸡结扎肠环模型研究荚梭菌毒力
Summary
在这里, 我们提出了一个在鸡小肠中手术创建 ‘ 肠结扎循环 ‘ 的协议。本程序允许在单个宿主中对多个荚菌株的毒力进行原位比较。这种方法明显地减少了通常需要的鸡的数量在类似的体内实验。
Abstract
采用各种体内感染模型对鸡进行坏死肠炎的研究。大多数这些使用的诱发因素的组合, 如球虫病和饮食, 与灌胃或管理通过饲料使用荚梭菌。在这些模型中, 多荚菌株对毒力研究的比较需要大量的主机来获得显著的结果。在研究过程中, 死亡率可能会很高, 这取决于实验模型, 因此在研究中提高了对动物福利的伦理关注。新的感染模型的开发需要较少的动物来研究发病机制, 但提供统计学上的显著和有效的结果, 在减少动物在研究中的使用是重要的。肠道结扎环模型已被用于研究梭菌感染的各种物种, 如小鼠, 兔和小牛。在手术过程中创建结扎环段, C. 荚菌株直接注入循环中, 在细菌和肠道粘膜之间建立密切接触。小肠和管腔的样本在程序终止后几个小时后采取。可以在每种动物中接种多个细菌菌株, 从而减少实验中所需的受试者数量。同时, 在全身麻醉下进行手术以减少动物疼痛。在鸡中, 这种模式比口服施用更适合荚菌株致病性, 因为需要较少的动物, 不需要诱发因素诱发疾病, 疼痛由止痛药控制。.鸡小肠结扎环模型的描述不充分, 标准化是其最佳使用的必要条件。这份手稿提供了所有必要的步骤, 以创建大量的肠道结扎循环在鸡和带来的关键点信息, 以获得有效的结果。
Introduction
使用动物模型来研究影响人类和动物的传染性疾病, 是评估控制特定疾病发病机制的毒力因素以及制定预防或治疗疾病的策略的中心工具1。尽管使用动物模型来研究各种疾病有许多优点, 但对这种做法的伦理关注却是产生的。研究人员需要尽量减少动物的使用, 同时获得显著和有效的结果。制定了3《 Rs 原则 (替换、减少和改进) 的概念, 以确保在这种审判中处理福利问题。在鸡坏死性肠炎研究中,体内鸡模型用于调查这种情况的病因、预防和治疗2、3、4、5。荚携带特定毒力因子, 如 NetB 毒素6的致病菌株, 被管理导致鸡坏死性肠炎7。因此, 替换原则在这种情况下很难实现, 因为这些毒力因子在其他动物物种中可能不那么重要。大多数鸡坏死性肠炎模型使用的危险因素的组合, 如球虫病和改变的饮食诱导坏死肠炎后, 灌胃的肉汤培养含有大量的C. 荚2.这些模型将导致大量的鸡的疾病, 死亡率可以是重要的在这种试验8, 从而引起关注的减少和细化原则在动物研究。
肠道结扎循环模型是研究肠道病原体引起的疾病的一种可取的替代方法, 以减少其原理和细化。在这些模型中, 被称为 ‘ 循环 ‘ 的肠道片段通过在肠道上放置连字来形成独立和密闭的隔间, 在这种情况下, 病原体可以单独注射9或与其他分子, 如疫苗候选10,11. 有兴趣的病原体与肠道细胞密切接触, 经过几个小时的感染时间, 可以恢复肠道样本以作进一步分析。这允许使用在同一动物的多个治疗和控制组。统计分析可以通过重复测量模型进行, 这增加了群体之间的歧视的力量, 减少了必要的鸡的数量相比口服灌胃试验。此外, 手术过程和随后的感染时间在持续全身麻醉和镇痛下进行, 从而尽量减少动物疼痛。闭环结扎是减少主机数量和创建更人性化的动物研究系统的理想模板。
肠道结扎循环模型在不同的物种, 如小牛, 兔子和小鼠2,9, 但在鸡7描述很差的描述。为了最有效地使用这个手术模型, 正确的技术和执行对于建立结扎的肠道循环, 以避免损害肠道完整性是必不可少的。本手稿的目的是描述一步一步的方法在创建在鸡模型的多个肠道循环。这种技术受外科医生的技能和经验的限制, 因为准确的程序对于项目的成功至关重要。
Protocol
Representative Results
Discussion
肠道循环模型已被描述在许多物种, 以研究寄主病原体相互作用和疾病的发病机制, 如梭菌荚, 梭杆菌和沙门氏菌起因7,9,13,14,15。它也被用来分析黏膜免疫反应16, 抗体的效果, 以消除肠道病原体排泄的细菌毒素11和评估疫…
Divulgations
The authors have nothing to disclose.
Acknowledgements
这项工作得到了蒙特利尔大学兽医学院家禽研究 (m. Boulianne) 主席的支持。Boulianne 和家长开发了模型。Boulianne 和家长进行了手术。伯恩斯博士协助制定了麻醉协议。家长博士编辑了这部影片并写了手稿。Drs 烧伤, Desrochers 和 Boulianne 编辑了手稿。
Materials
| EZ-Scrub 747 | Becton Dickinson and Company | 4% chlorhexidine gluconate detergent impregnated sterile brush. No catalog number available. Web address: https://www.bd.com/en-us/offerings/capabilities/infection-prevention/surgical-hand-scrubs/ez-scrub-preoperative-surgical-scrub-brushes | |
| Isopropyl alcohol 70% USP 4 L | Commercial isopropyl alcohol | P016IP70 | Web address: http://www.comalc.com/products/ |
| Chlorexidine | Sigma-Aldrich | 282227-1G | Chlorhexidine gluconate solution, must be diluted to 4%. Web address: http://www.sigmaaldrich.com/catalog/product/aldrich/282227?lang=fr®ion=CA&gclid=EAIaIQobChMI8vHr4_OY1wIV3rrACh2ZWQKuEAAYAiAAEgLKx_D_BwE |
| Surgical Drape Small (27 inch x 24 inch) with 3 x 6 inch Fenestration | Veterinary Specialty Products | 32724 | Web address: http://www.vetspecialtyproducts.com/index.cfm?fuseaction=ecommercecatalog.detail&productgroup_id=15 |
| Scalpel blades #3 | Swann-Morton | 301 | Web address: https://www.swann-morton.com/product/16.php |
| Sterile saline NaCl 0.9% | Sigma-Aldrich | S8776-100ML | Web address: http://www.sigmaaldrich.com/catalog/product/sigma/s8776?lang=fr®ion=CA |
| Vicryl 3-0 | Ethicon | D9003 | Polyglactin multifilament absorbable suture material. Web address: http://www.ethicon.com/healthcare-professionals/products/wound-closure/absorbable-sutures/coated-vicryl-polyglactin-910-suture |
| Syringe 1 ml with 26G needle | Becton Dickinson and Company | 329652 | Web address: https://www.bd.com/en-us/offerings/capabilities/diabetes-care/insulin-syringes/bd-1-ml-conventional-insulin-syringes |
| Brain Heart Infusion | Sigma-Aldrich | 1104930500 | Web address: http://www.sigmaaldrich.com/catalog/product/mm/110493?lang=fr®ion=CA&cm_sp=Insite-_-prodRecCold_xorders-_-prodRecCold2-1 |
| Eppendorf tube | Sigma-Aldrich | T9661-500EA | Microcentrifuge tube. Web address: http://www.sigmaaldrich.com/catalog/product/sigma/t9661?lang=fr®ion=CA&gclid=EAIaIQobChMI-YizzfiY1wIVRkCGCh30SQjuEAAYAiAAEgLK5fD_BwE |
| Formalin solution, buffered neutral, 10% | Sigma-Aldrich | HT501128-4L | Ratio tissue : formalin of 1: 10 for adequate fixation. Web address: http://www.sigmaaldrich.com/catalog/product/sigma/ht501128?lang=fr®ion=CA |
| Clostridium perfringens strains | Université de Montréal, Chaire en recherche avicole | N/A | Specific to each laboratory, available upon request to correspondant author |
References
- Vitale, A., Chiarotti, F., Alleva, E. The use of animal models in disease research. Rare diseases and orphan drugs. 2 (1), 1-4 (2015).
- Uzal, F. A., et al. Animal models to study the pathogenesis of human and animal Clostridium perfringens infections. Veterinary Microbiology. , (2015).
- Bernier, G., Phaneuf, J. B., Filion, R. Necrotic enteritis in broiler chickens. III. Study of the factors favoring the multiplication of Clostridium perfringens and the experimental transmission of the disease. Canadian Journal of Comparative Medicine. 41 (1), 112-116 (1977).
- Al-Sheikhly, F., Truscott, R. B. The pathology of necrotic enteritis of chickens following infusion of broth cultures of Clostridium perfringens into the duodenum. Avian Diseases. 21 (2), 230-240 (1977).
- Wicker, D. L., Iscrigg, W. N., Trammell, J. H. The control and prevention of necrotic enteritis in broilers with zinc bacitracin. Poultry Science. 56 (4), 1229-1231 (1977).
- Keyburn, A. L., et al. NetB, a new toxin that is associated with avian necrotic enteritis caused by Clostridium perfringens. PLoS Pathog. 4 (2), e26 (2008).
- Timbermont, L., et al. Origin of Clostridium perfringens isolates determines the ability to induce necrotic enteritis in broilers. Comp Immunol Microbiol Infect Dis. 32 (6), 503-512 (2009).
- Paradis, M. A., et al. Efficacy of avilamycin for the prevention of necrotic enteritis caused by a pathogenic strain of Clostridium perfringens in broiler chickens. Avian Pathol. 45 (3), 365-369 (2016).
- Valgaeren, B., et al. Lesion development in a new intestinal loop model indicates the involvement of a shared Clostridium perfringens virulence factor in haemorrhagic enteritis in calves. Journal of Comparative Pathology. 149 (1), 103-112 (2013).
- Goossens, E., et al. The C-terminal domain of Clostridium perfringens alpha toxin as a vaccine candidate against bovine necrohemorrhagic enteritis. Vet Res. 47 (1), 52 (2016).
- Goossens, E., et al. Toxin-neutralizing antibodies protect against Clostridium perfringens-induced necrosis in an intestinal loop model for bovine necrohemorrhagic enteritis. BMC Vet Res. 12 (1), 101 (2016).
- Parent, E., Archambault, M., Charlebois, A., Bernier-Lachance, J., Boulianne, M. A chicken intestinal ligated loop model to study the virulence of Clostridium perfringens isolates recovered from antibiotic-free chicken flocks. Avian Pathol. 46 (2), 138-149 (2017).
- Janvilisri, T., et al. Temporal differential proteomes of Clostridium difficile in the pig ileal-ligated loop model. PLoS One. 7 (9), e45608 (2012).
- Aabo, S., et al. Development of an in vivo model for study of intestinal invasion by Salmonella enterica in chickens. Infection and Immunity. 68 (12), 7122-7125 (2000).
- Meurens, F., et al. Early immune response following Salmonella enterica subspecies enterica serovar Typhimurium infection in porcine jejunal gut loops. Vet Res. 40 (1), 5 (2009).
- Gerdts, V., et al. Multiple intestinal ‘loops’ provide an in vivo model to analyse multiple mucosal immune responses. J Immunol Methods. 256 (1-2), 19-33 (2001).
- Wade, B., et al. The adherent abilities of Clostridium perfringens strains are critical for the pathogenesis of avian necrotic enteritis. Vet Microbiol. 197, 53-61 (2016).
- Cooper, K. K., et al. Virulence for chickens of Clostridium perfringens isolated from poultry and other sources. Anaerobe. 16 (3), 289-292 (2010).
- Cooper, K. K., Songer, J. G. Virulence of Clostridium perfringens in an experimental model of poultry necrotic enteritis. Veterinary Microbiology. 142 (3-4), 323-328 (2010).
- Chalmers, G., Bruce, H. L., Toole, D. L., Barnum, D. A., Boerlin, P. Necrotic enteritis potential in a model system using Clostridium perfringens isolated from field outbreaks. Avian Diseases. 51 (4), 834-839 (2007).
