肉鸡弯曲杆菌噬菌体的筛选及应用

doi:10.16431/j.cnki.1671-7236.2021.09.018

摘要/Abstract

摘要： 试验旨在筛选肉鸡弯曲杆菌特异性裂解型噬菌体并对其在肉鸡生产中的合理应用进行探讨。从肉鸡屠宰场收集弯曲杆菌噬菌体筛选样本，通过与当地肉鸡弯曲杆菌流行株共孵育、增殖、纯化获得特异性弯曲杆菌噬菌体。对上述弯曲杆菌噬菌体生物学特性（宿主范围、致死曲线、形态学特征等）进行鉴定；将筛选得到的弯曲杆菌噬菌体作为饲料添加剂，以5×10⁷、1×10⁸、5×10⁸、1×10⁹和5×10⁹ PFU/d 5个添加量添加于38日龄雄性罗斯308肉鸡饲粮中，观察不同噬菌体添加量对肉鸡盲肠内容物中弯曲杆菌的清除效果；最后通过比较2种噬菌体不同配比条件对肉鸡弯曲杆菌清除率及对肉鸡生长性能的影响建立使用方案。结果显示，试验从当地肉鸡屠宰场以空肠弯曲杆菌标准株L26为宿主菌共分离到12株空肠弯曲杆菌噬菌体，与从肉鸡养殖场分离的7株空肠弯曲杆菌共孵育，并选择裂解谱广且噬菌斑大的噬菌体BP11和BP12进行纯化和增殖。经形态学鉴定，2株噬菌体均符合肌尾噬菌体科（Myoviridae）特征，并具有较宽的裂解谱，但二者对相同宿主菌的裂解能力表现差异性；BP11添加量为1×10⁹ PFU/d，BP12添加量为5×10⁸ PFU/d时均能达到显著裂解效果；BP11与BP12混合添加组在不影响屠宰指标的前提下可显著降低肉鸡泄殖腔弯曲杆菌携带量。本研究结果为肉鸡生产过程中弯曲杆菌污染的防控及噬菌体的合理利用奠定理论基础。

关键词: 肉鸡; 弯曲杆菌; 噬菌体; 筛选; 应用

Abstract: The aim of the experiment was to screen out the lytic bacteriophages of Campylobacter and to explore their rational application in broiler production. Campylobacter bacteriophage screening samples were collected from broiler slaughterhouse, and specific Campylobacter bacteriophages were obtained by co-incubating, proliferating and purifying with local Campylobacter epidemic strains. The biological characteristics (host range, lethal curve, morphological characteristics, etc.) of Campylobacter bacteriophage were identified. As a feed additive, it was added to the diets of 38-day-old male Ross 308 broilers with 5×10⁷, 1×10⁸, 5×10⁸, 1×10⁹ and 5×10⁹ PFU/d 5 different amounts to observe the scavenging effect of Campylobacter in cecal contents of broilers. Finally, by comparing the effects of different phage ratio on the clearance rate of Campylobacter and the growth performance of broilers, the application scheme was established. The results showed that a total of 12 strains of Campylobacter jejuni bacteriophages were isolated from local broiler slaughterhouses using Campylobacter jejuni L26 as host bacteria. The bacteriophages BP11 and BP12 which had wide lytic spectrum and large plaque were chose by co-incubating, purifying and proliferating with 7 strains of Campylobacter jejuni from broiler farms. By morphological identification, the two bacteriophages were in accordance with the characteristics of Myoviridae and had a wide lytic spectrum, but their lytic ability of the same host bacteria was different. The bactericidal effect was significant when the BP11 and BP12 addition amount were 1×10⁹ and 5×10⁸ PFU/d, respectively. Bacteriopages BP11 and BP12 mixed addition group could significantly reduce the carrying rate of Campylobacter in broiler production without affecting the slaughter index. These results provided theoretical foundation for the prevention and control of Campylobacter contamination and the rational utilization of bacteriophages in broilers.

Key words: broiler; Campylobacter; bacteriophage; screening; application

中图分类号:

S858.31

席丽, 秦新喜, 孙新峰, 宋毓民, 李志强. 肉鸡弯曲杆菌噬菌体的筛选及应用[J]. 中国畜牧兽医, 2021, 48(9): 3273-3282.

XI Li, QIN Xinxi, SUN Xinfeng, SONG Yumin, LI Zhiqiang. Screening and Application of Campylobacter Bacteriophage in Broilers[J]. China Animal Husbandry and Veterinary Medicine, 2021, 48(9): 3273-3282.

参考文献

[1] LU T, MARMION M, FERONE M, et al. On farm interventions tominimise Campylobacter spp.contamination in chicken[J]. British Poultry Science, 2021, 62(1):53-67.
[2] THAMES H T, SUKUMARAN A T.A review of Salmonella and Campylobacter in broiler meat:Emerging challenges and food safety measures[J]. Foods, 2020, 9(6):e776.
[3] 黄亚奇, 熊静禹, 沈达, 等.空肠弯曲杆菌毒力因子及其致病机制研究进展[J]. 中国家禽, 2019, 41(22):46-51. HUANG Y Q, XIONG J Y, SHEN D, et al. Advances in research on virulence factors and pathogenic mechanisms of Campylobacter jejuni[J]. China Poultry, 2019, 41(22):46-51.(in Chinese)
[4] HANSSON I, SANDBERG M, HABIB I, et al. Knowledge gaps in control of Campylobacter for prevention of campylobacteriosis[J]. Transboundary and Emerging Diseases, 2018, 65(1):30-48.
[5] ABE H, KOYAMA K, KOSEKI S.Modeling invasion of Campylobacter jejuni into human small intestinal epithelial-like cells by Bayesian inference[J]. Applied and Environmental Microbiology, 2020, 87(1):e01551-20.
[6] BARAKAT A M A, EI-RAZIK K A A, EIFADALY H A, et al. Prevalence, molecular detection, and virulence gene profiles of Campylobacter species in humans and foods of animal origin[J]. Veterinary World, 2020, 13(7):1430-1438.
[7] PEREZ-BOTO D, HERRERA-LEON S, GARCIA-PENA F J, et al. Molecular mechanisms of quinolone, macrolide, and tetracycline resistance among Campylobacter isolates from initial stages of broiler production[J]. Avian Pathology, 2014, 43(2):176-182.
[8] VOHRA P, CHINTOAN-UTA C, TERRA V S, et al. Evaluation of glycosylated FlpA and SodB as subunit vaccines against Campylobacter jejuni colonisation in chickens[J]. Vaccines, 2020, 8(3):520.
[9] PEH E, KITTLER S, REICH F, et al. Antimicrobial activity of organic acids against Campylobacter spp.and development of combinations-A synergistic effect?[J]. PLoS One, 2020, 15(9):e0239312.
[10] SEFCOVA M, LARREA-ÁLVAREZ M, LARREA-ÁLVAREZ C, et al. Effects of Lactobacillus fermentum supplementation on body weight and pro-inflammatory cytokine expression in Campylobacter jejuni-challenged chickens[J]. Veterinary Sciences, 2020, 7(3):121.
[11] WAGENBERG C P A, HORNE P L M, ASSELDONK M A P M, et al. Cost-effectiveness analysis of using probiotics, prebiotics, orsynbiotics to control Campylobacter in broilers[J]. Poultry Science, 2020, 99(8):4077-4084.
[12] PITTER J G, VOKO Z, JOZWIAK A, et al. Campylobacter control measures in indoor broiler chicken:Critical re-assessment of cost-utility and putative barriers to implementation[J]. Epidemiology and Infection, 2018, 146(11):1433-1444.
[13] HOOTON S, D'ANGELANTONIO D, HU Y, et al. Campylobacter bacteriophage DA10:An excised temperate bacteriophage targeted by CRISPR-cas[J]. BMC Genomics, 2020, 21(1):400.
[14] SAMARTH D P, KWON Y M.Horizontal genetic exchange of chromosomally encoded markers between Campylobacter jejuni cells[J]. PLoS One, 2020, 15(10):e0241058.
[15] RIVERA-MENDOZA D, MARTINEZ-FLORES I, SANTAMARIA R I, et al. Genomic analysis reveals the genetic determinants associated with antibiotic resistance in the zoonotic pathogen Campylobacter spp.distributed globally[J]. Frontiers in Microbiology, 2020, 11:513070.
[16] SEO H, DUAN Q, ZHANG W P, et al. Vaccines against gastroenteritis, current progress and challenges[J]. Gut Microbes, 2020, 11(6):1486-1517.
[17] 魏炳栋, 丛聪, 李淑英, 等.噬菌体在防控畜禽细菌性感染中的应用[J]. 中国畜牧兽医, 2020, 47(1):190-200. WEI B D, CONG C, LI S Y, et al. Application of bacteriophage to prevent and control bacterial infection in livestock and poultry[J]. China Animal Husbandry & Veterinary Medicine, 2020, 47(1):190-200.(in Chinese)
[18] RICHARDS P J, CONNERTON P L, CONNERTON I F, et al. Phagebiocontrol of Campylobacter jejuni in chickens does not produce collateral effects on the gut microbiota[J]. Frontiers in Microbiology, 2019, 10:476.
[19] ADIGUZEL M C, SIGIRCI B D, CELIK B, et al. Phenotypic and genotypic examination of antimicrobial resistance in thermophilic Campylobacter species isolated from poultry in Turkey[J]. Journal of Veterinary Research, 2018, 62(4):463-468.
[20] FURUTA M, NASU T, UMEKI K, et al. Characterization and application of lytic bacteriophages against Campylobacter jejuni isolated from poultry in Japan[J]. Biocontrol Science, 2017, 22(4):213-221.
[21] CARVALHO C M, GANNON B W, HALFHIDE D E, et al. The in vivo efficacy of two administration routes of a phage cocktail to reduce numbers of Campylobacter coli and Campylobacter jejuni in chickens[J]. BMC Microbiology, 2010, 10:232.
[22] CHINIVASAGAM H N, ESTELLA W, MADDOCK L, et al. Bacteriophages to control Campylobacter in commercially farmed broiler chickens, in Australia[J]. Frontiers in Microbiology, 2020, 11:632.
[23] SØRENSEN M C H, GENCAY Y E, BIRK T, et al. Primary isolation strain determines both phage type and receptors recognised by Campylobacter jejuni bacteriophages[J]. PLoS One, 2015, 10(1):e0116287.
[24] ROSENQUIST H, SOMMER H M, NIELSEN N L, et al. The effect of slaughter operations on the contamination of chicken carcasses with thermotolerant Campylobacter[J]. International Journal of Food Microbiology, 2006, 108(2):226-232.
[25] USHANOV L, LASAREISHVILI B, JANASHIA I, et al. Application of Campylobacter jejuni phages:Challenges and perspectives[J]. Animals, 2020, 10(2):279.