基于16SrDNA高通量测序技术分析受水灾影响猪场水样的菌群特征

doi:10.11843/j.issn.0366-6964.2023.05.030

摘要/Abstract

摘要： 2021年7月,河南省部分地区突发特大水灾,致使受灾区域内的猪场损失惨重,本研究旨在探究水灾发生后猪场水样中存在的病原菌,为猪场防控可能出现的疾病提供技术支撑。通过实地采样和委托采样的方式,获得了26个受灾猪场中的48份水样,包括19份猪饮用水、22份场内积雨水和7份废弃污水,采用菌落计数和高通量测序技术,对样品的细菌总数和菌群丰度进行分析研究,同时对采集的水样开展消毒剂杀菌试验。结果发现:饮用水、积雨水和污水中的平均含菌量分别为1.55×10⁵、1.88×10⁸和3.70×10⁹ CFU·mL^-1,其中污水中的细菌总数最多,其次为积雨水,饮用水中的细菌总数最少,但远超国家饮用水的安全标准。本研究在不同种类的水样中均检测到了链球菌属、埃希氏杆菌属、弓形杆菌属、不动杆菌属和假单胞菌属等潜在的致病菌属,以及嗜低温弓形杆菌、猪链球菌、大肠杆菌、鲍曼不动杆菌和豚鼠气单胞菌等致病菌。杀菌试验显示,氯制剂终浓度达到2 500mg·L^-1,作用30 min即可杀灭积雨水和污水中所有的细菌。本研究发现,水灾后养猪场内的水样中含有多种致病菌,规模化猪场可选用含氯消毒剂对场内污染的水体进行消杀,以确保人和猪群的健康安全。

关键词: 16S rDNA, 猪场, 洪水, 细菌

Abstract: In July 2021, a severe flood occurred in some area of Henan Province, pig farms in these areas suffered heavy losses. The purpose of the current research is to explore the bacteria in the flood-affected water samples of pig farms, so as to provide technical support for the prevention and control of possible diseases. Different types of water samples including 19 drinking water, 22 rainwater, and 7 waste sewage samples were collected by field sampling and airmail from 26 farms in the affected areas. Using colony counting and high-throughput sequencing technology, the total number and the abundance of bacteria in the collected samples were analyzed, and disinfection tests were carried out for the collected water samples. The results showed that the total bacterial counts in drinking water, rainwater and sewage were 1.55×10⁵, 1.88×10⁸and 3.70×10⁹ CFU·mL^-1, respectively. The total number of bacteria in the sewage was the highest, followed by the rainwater, and that of the drinking water was the lowest, but still higher than the national standard of drinking water. By mean of high-throughput detection technology, potential pathogenic genus such as Streptococcus, Escherichia, Arcobacter, Actinobacter and Pseudomonas, as well as waterborne pathogenic bacteria such as Arcobacter cryaerophilus, Streptococcus suis, Escherichia coli, Acinetobacter baumannii and Aeromonas caviae, were detected in water samples. Bactericidal tests showed that when the final concentration of chlorine reaches 2 500 mg·L^-1, all bacteria in rainwater and sewage were killed within 30 minutes. It was demonstrated that water samples in pig farms after floods contained a variety of pathogenic bacteria and pig farms can choose chlorine-containing disinfectants to kill the bacteria in polluted water in the farm to ensure the health of humans and pigs.

Key words: 16S rDNA, pig farm, flood, bacteria

中图分类号:

S855.1

洪勃, 孙琪, 李栋凡, 于学祥, 库旭钢, 何启盖. 基于16SrDNA高通量测序技术分析受水灾影响猪场水样的菌群特征[J]. 畜牧兽医学报, 2023, 54(5): 2092-2100.

HONG Bo, SUN Qi, LI Dongfan, YU Xuexiang, KU Xugang, HE Qigai. Investigation of Bacterial Communities in Water Samples from Flood-affected Pig Farms Using 16S rDNA High-throughput Sequencing Technology[J]. Acta Veterinaria et Zootechnica Sinica, 2023, 54(5): 2092-2100.

参考文献 26

[1]	郭又春,徐利,卢纪和,等.北京地区规模化猪场猪饮用水细菌污染情况的调查与分析[J].中国猪业,2017,12(3):77-79.GUO Y C,XU L,LU J H,et al.Investigation and analysis of bacterial contamination in drinking water of pigs from large-scale pig farms in Beijing[J]. China Swine Industry,2017,12(3):77-79.(in Chinese)
[2]	李建波,陈宏达,耿鹤鸣,等.安徽地区规模化猪场猪饮用水细菌污染情况的调查与分析[J].猪业科学,2018,35(2):98-99.LI J B,CHEN H D,GENG H M,et al.Investigation and analysis of bacterial contamination in drinking water of pig farms in Anhui region[J]. Swine Industry Science,2018,35(2):98-99.(in Chinese)
[3]	阎宏,李晓梅,宋彦芳,等.养猪场空气和水体污染状况调查[J].畜牧与兽医,2003,35(10):28-29.YAN H,LI X M,SONG Y F,et al.Investigation of air and water pollution in pig farms[J]. Animal Husbandry & Veterinary Medicine,2003,35(10):28-29.(in Chinese)
[4]	中华人民共和国卫生部,中国国家标准化管理委员会.GB 5749-2006生活饮用水卫生标准[S].北京:中国标准出版社,2007.Ministry of Health of the People's Republic of China,China National Standardization Administration.GB 5749-2006 Standards for drinking water quality[S].Beijing:Standards Press of China,2007.(in Chinese)
[5]	MILAKOVIĆ M,VESTERGAARD G,GONZÁLEZ-PLAZA J J,et al.Pollution from azithromycin-manufacturing promotes macrolide-resistance gene propagation and induces spatial and seasonal bacterial community shifts in receiving river sediments[J]. Environ Int,2019,123:501-511.
[6]	EL-CHAKHTOURA J,PREST E,SAIKALY P,et al.Dynamics of bacterial communities before and after distribution in a full-scale drinking water network[J]. Water Res,2015,74:180-190.
[7]	王占生,刘文君.微污染水源饮用水处理[M].北京:中国建筑工业出版社,1999.WANG Z S,LIU W J.Treatment of drinking water from slightly polluted sources[M].Beijing:China Architecture & Building Press,1999.(in Chinese)
[8]	孙健龙,张素梅,管晓芸,等.机械通气致下呼吸道黄杆菌属感染的临床研究[J].中华医院感染学杂志,2012,22(11):2420-2422.SUN J L,ZHANG S M,GUAN X Y,et al.A clinical study on lower respiratory tract infections caused by Flavobacterium after mechanical ventilation[J]. Chinese Journal of Nosocomiology,2012,22(11):2420-2422.(in Chinese)
[9]	LIN C J,WADE T J,HILBORN E D.Flooding and Clostridium difficile infection:a case-crossover analysis[J]. Int J Environ Res Public Health,2015,12(6):6948-6964.
[10]	ALAM M,ISLAM A,BHUIYAN N A,et al.Clonal transmission,dual peak,and off-season cholera in Bangladesh[J]. Infect Ecol Epidemiol,2011,1(1):7273.
[11]	毕水莲,孟赫诚.致病性弓形杆菌属生物学特性及诊断研究进展[J].现代食品科技,2013,29(1):211-214.BI S L,MENG H C.Biological characteristics and diagnoses of pathogenic arcobacter species[J]. Modern Food Science and Technology,2013,29(1):211-214.(in Chinese)
[12]	COLLADO L,FIGUERAS M J.Taxonomy,epidemiology,and clinical relevance of the genus Arcobacter[J]. Clin Microbiol Rev,2011,24(1):174-192.
[13]	KRISTENSEN J M,NIERYCHLO M,ALBERTSEN M,et al.Bacteria from the genus Arcobacter are abundant in effluent from wastewater treatment Plants[J]. Appl Environ Microbiol,2020,86(9):e03044-19.
[14]	RIZZO L,MANAIA C,MERLIN C,et al.Urban wastewater treatment plants as hotspots for Antibiotic resistant bacteria and genes spread into the environment:a review[J]. Sci Total Environ,2013,447:345-360.
[15]	DE OLIVEIRA S J,BAETZ A L,WESLEY I V,et al.Classification of Arcobacter species isolated from aborted pig fetuses and sows with reproductive problems in Brazil[J]. Vet Microbiol,1997,57(4):347-354.
[16]	DE OLIVERIA S J,WESLEY I V,BAETZ A L,et al. Arcobacter cryaerophilus and Arcobacter butzleri isolated from preputial fluid of boars and fattening pigs in Brazil[J]. J Vet Diagn Invest,1999,11(5):462-464.
[17]	LEHNER A,TASARA T,STEPHAN R.Relevant aspects of Arcobacter spp.as potential foodborne pathogen[J]. Int J Food Microbiol,2005,102(2):127-135.
[18]	高国强,李树东,王珊珊,等.流产胎猪鲍曼不动杆菌的分离鉴定[J].中国兽医科学,2017,47(12):1577-1582.GAO G Q,LI S D,WANG S S,et al.Isolation and identification of Acinetobacter baumannii from abortion fetal pigs[J]. Chinese Veterinary Science,2017,47(12):1577-1582.(in Chinese)
[19]	邱立,王兴龙,郝华芳,等.仔猪鲍曼不动杆菌的分离鉴定及生物学特性研究[J].中国预防兽医学报,2012,34(11):882-885.QIU L,WANG X L,HE H F,et al.Isolation characteristics and of Acinetobaeter baumannii from diarrhea piglets[J]. Chinese Journal of Preventive Veterinary Medicine,2012,34(11):882-885.(in Chinese)
[20]	李任文,李峥,黄武军,等.1例豚鼠气单胞菌致感染性休克死亡病例报道[J].中国现代医生,2015,53(11):143-145.LI R W,LI Z,HUANG W J,et al.A case report of infectious shock death caused by Aeromonas in guinea pig[J]. China Modern Doctor,2015,53(11):143-145.(in Chinese)
[21]	陈枝华.仔猪白痢混合感染豚鼠气单胞菌死亡病例的诊断报告[J].中国畜禽传染病,1993,(1):18-19.CHEN Z H.Diagnostic report of death cases of piglet mixed dysentery infection with guinea pig Aeromonas[J]. Chinese Journal of Preventive Veterinary Medicine,1993,(1):18-19.(in Chinese)
[22]	韩桂英,张淑滨,杨继军,等.一起因食猪排骨引起的41人豚鼠气单胞菌食物中毒的报告[J].中国食品卫生杂志,1992,4(3):35-36.HAN G Y,ZHANG S B,YANG J J,et al.A report of 41 human guinea-pig Aeromonas food poisoning caused by eating pork ribs[J]. Chinese Journal of Food Hygiene,1992,4(3):35-36.(in Chinese)
[23]	LUN Z R,WANG Q P,CHEN X G,et al. Streptococcus suis:an emerging zoonotic pathogen[J]. Lancet Infect Dis,2007,7(3):201-209.
[24]	ZUBERBUHLER B,CARIFI G. Pseudomonas putida infection of the conjunctiva[J]. Infection,2012,40(5):579-580.
[25]	ROSCHANSKI N,HADZIABDIC S,BOROWIAK M,et al.Detection of VIM-1-producing Enterobacter cloacae and Salmonella enterica serovars infantis and goldcoast at a breeding pig farm in Germany in 2017 and their molecular relationship to former VIM-1-Producing S.infantis isolates in German livestock production[J]. Clin Sci Epidemiol,2019,4(3):e00089-19.
[26]	FARD R M N,HEUZENROEDER M W,BARTON M D.Antimicrobial and heavy metal resistance in commensal enterococci isolated from pigs[J]. Vet Microbiol,2011,24(2-4):276-282.