摘要

       畜禽舍内微生物气溶胶中含有大量的病原菌、病毒、真菌等对人体有害的微生物以及抗生素抗性基因,其可以通过空气将致病因子传出舍内并在外界环境中快速、广泛地传播,对人类和其他动物健康造成危害。目前,对不同畜禽舍内气溶胶中的菌群结构及抗生素污染情况差异性研究的报道较少。掌握不同畜禽舍内气溶胶中菌群结构特征及抗生素抗性基因污染情况,是有针对性的解决不同动物饲养场空气污染及疾病防控问题的重要前提。因此,本研究将以三种畜禽饲养场共计12个养殖舍(4个猪舍,4个鸡舍,4个牛舍)内空气样本为研究对象,利用传统培养法分析不同畜禽舍内气载需氧菌、气载真菌浓度及粒径分布情况;利用高通量测序技术分析不同畜禽舍内气溶胶样本中菌群结构特征、病原菌含量;利用普通PCR和qPCR技术分析不同畜禽舍气溶胶样本中抗生素抗性基因及整合子污染情况。实验结果表明,鸡舍内微生物浓度较高,气载需氧菌浓度为7.514×10~3CFU/m~3~2.313×10~4 CFU/m~3;其次为猪舍和牛舍,分别为8.621×10~3CFU/m~3~14.579×10~3 CFU/m~3、1.805×10~3CFU/m~3~3.243×10~3 CFU/m~3。气载真菌浓度鸡舍内含量最高,为1.813×10~3 CFU/m~3~7.550×10~3 CFU/m~3,其次为猪舍和牛舍,分别为1.401×10~3 CFU/m~3~5.441×10~3 CFU/m~3、1.752×10~3 CFU/m~3~2.367×10~3CFU/m~3。三种畜禽舍内气载需氧菌主要分布在Ⅰ~Ⅰ层级上,分别约占79.85%(猪舍)、73.79%(鸡舍)、68.63%(牛舍),空气动力学直径大于3.3μm。而气载真菌分布情况各不相同,猪舍内气载真菌主要分布在Ⅰ层级,约占43.04%,其空气动力学直径大于3.3μm;鸡舍中主要分布在Ⅱ、Ⅰ层级上,约占总数的51.61%,其空气动力学直径3.3~7.0μm;而在牛舍中,粒子主要分布在Ⅰ、Ⅴ层级上,约占52.52%,其空气动力学直径为1.1~4.7μm。通过高通量测序后共获得408996个有效Tags,通过注释及聚类分析共获得33个菌门和614个菌属。其中厚壁菌门(Firmicutes)、放线菌门(Actinobacteria)、变形菌门(Proteobacteria)以及拟杆菌门(Bacteroidetes)为主要优势菌门。粪杆菌属(Faecalibacterium)、链霉菌属(Streptomyces)、小单胞菌属(Micromonospora)为鸡舍内主要的优势菌属;梭状芽孢杆菌属(Clostridium_sensu_stricto_1)、乳酸菌(Lactobacillus)、Terrisporobacter为猪舍内主要优势菌属;疣微菌属(Ruminococcaceae)、不动杆菌属(Acinetobacter)、泛生菌属(Pantoea)为牛舍内主要的优势菌属,其相对丰度在不同畜禽舍内气溶胶样本中存在着明显的差异性。同时还发现不同畜禽舍内病原菌含量也存在一定的差异性,其中在猪舍气溶胶样本中病原菌含量最高,达到19.53%,其次为牛舍达到12.06%,鸡舍内含量最低为4.22%。此外,畜禽舍内的悬浮颗粒浓度及有害气体浓度与气溶胶中微生物菌群多样之间存在明显的相关性。通过普通PCR对畜禽舍内气溶胶样本中8种四环素类抗性基因及一类整合子进行定性分析,结果发现tetG、tetM、tetO、tetQ、tetW、tetZ以及intI1在三种畜禽舍内均有检出,tetA只在鸡舍中检出,tetB只在猪舍中检出。通过qPCR技术对tetG、tetM、tetO、tetQ、tetW、tetZ以及intI 1进行定量分析,结果发现其丰度在不同畜禽舍内存在着显著的差异性。四环素类抗性基因在猪舍内含量为7.52×10~2~1.75×10~6 copies/ng·DNA;牛舍内为1.11×10~3~2.02×10~5 copies/ng·DNA;鸡舍内为3.29×10~3~7.68×10~5 copies/ng·DNA。一类整合子(intI1)含量猪舍最高6.24×10~4 copies/ng·DNA;其次为鸡舍2.78×10~4 copies/ng·DNA;牛舍含量最低为1.30×10~4 copies/ng·DNA。畜禽养殖舍内微生物气溶胶可作为一个病原菌及抗性基因的存储库。由于不同畜禽舍空气中病原菌浓度及抗性基因污染情况存在着显著的差异。因此,针对畜禽养殖类型的不同而采用不同的疾病防控手段以及环境控制手段来降低养殖环境对周围环境造成的污染具有十分重要的意义。

       Microbial aerosols in livestock and poultry houses contain a large number of pathogenic bacteria, viruses, fungi and other harmful microorganisms and antibiotic resistance genes, which can pass pathogenic factors out of the house through the air and spread quickly and widely in the external environment , Causing harm to the health of humans and other animals. At present, there are few reports on the differences of the microbial structure and antibiotic pollution in aerosols in different livestock and poultry houses. To master the characteristics of the bacterial flora in aerosols in different livestock and poultry houses and the contamination of antibiotic resistance genes is an important prerequisite for targeted solutions to the problems of air pollution and disease prevention and control in different animal farms. Therefore, this study will take the air samples in 12 breeding houses (4 pig houses, 4 chicken houses, and 4 cow houses) in three kinds of livestock and poultry farms as the research object, and use traditional culture methods to analyze the air samples in different livestock and poultry houses. Concentration and particle size distribution of airborne aerobes and airborne fungi; use high-throughput sequencing technology to analyze the characteristics of bacterial flora and pathogen content in aerosol samples in different livestock and poultry houses; use ordinary PCR and qPCR techniques to analyze different livestock and poultry Contamination of antibiotic resistance genes and integrons in aerosol samples from the house. The experimental results show that the concentration of microorganisms in the chicken house is relatively high, and the concentration of airborne aerobic bacteria is 7.514×10~3CFU/m~3~2.313×10~4 CFU/m~3; the second is the pig house and the cow house, which are 8.621 ×10~3CFU/m~3~14.579×10~3 CFU/m~3, 1.805×10~3CFU/m~3~3.243×10~3 CFU/m~3. The airborne fungus concentration is the highest in the chicken house, which is 1.813×10~3 CFU/m~3~7.550×10~3 CFU/m~3, followed by the pig house and cow house, which are 1.401×10~3 CFU/m respectively ~3~5.441×10~3 CFU/m~3, 1.752×10~3 CFU/m~3~2.367×10~3CFU/m~3. The airborne aerobic bacteria in the three kinds of livestock and poultry houses are mainly distributed on the I~I level, accounting for about 79.85% (pig house), 73.79% (chicken house), 68.63% (cow house), and the aerodynamic diameter is greater than 3.3 μm. The distribution of airborne fungi is different. Airborne fungi in pig houses are mainly distributed on level I, accounting for about 43.04%, and their aerodynamic diameter is greater than 3.3 μm; in chicken houses, they are mainly distributed on level II and I, accounting for about 43.04%. 51.61% of the total, its aerodynamic diameter is 3.3~7.0μm; while in the cowshed, the particles are mainly distributed on the I and V levels, accounting for about 52.52%, and its aerodynamic diameter is 1.1~4.7μm. After high-throughput sequencing, a total of 408996 valid tags were obtained, and a total of 33 phyla and 614 genera were obtained through annotation and cluster analysis. Among them, Firmicutes, Actinobacteria, Proteobacteria and Bacteroidetes are the main phyla. Faecalis (Faecalibacterium), Streptomyces (Streptomyces), and Micromonospora (Micromonospora) are the main dominant genera in the chicken house; Clostridium_sensu_stricto_1, Lactobacillus (Lactobacillus), and Terrisporobacter are the pig houses The main dominant bacterial genera in the cattle house; Ruminococcaceae, Acinetobacter, and Pantoea are the main dominant bacterial genera in the cattle house, and their relative abundance is in different livestock and poultry houses. There are obvious differences in the sol samples. At the same time, it was also found that there are some differences in the pathogenic bacteria content in different livestock and poultry houses. Among them, the pathogenic bacteria content in the pig house aerosol samples is the highest, reaching 19.53%, followed by the cattle house with 12.06%, and the chicken house with the lowest content of 4.22%. In addition, there is an obvious correlation between the concentration of suspended particles and harmful gas in livestock and poultry houses and the diversity of microbial flora in aerosols. Qualitative analysis of 8 tetracycline resistance genes and one type of integron in aerosol samples in livestock and poultry houses by ordinary PCR showed that tetG, tetM, tetO, tetQ, tetW, tetZ, and intI1 were found in the three types of livestock and poultry houses. All were detected, tetA was only detected in the chicken house, and tetB was only detected in the pig house. Quantitative analysis of tetG, tetM, tetO, tetQ, tetW, tetZ and intI 1 by qPCR technology revealed that their abundances are significantly different in different livestock and poultry houses. The content of tetracycline resistance genes in the pig house is 7.52×10~2~1.75×10~6 copies/ng·DNA; in the cow house, it is 1.11×10~3~2.02×10~5 copies/ng·DNA; chicken The house is 3.29×10~3~7.68×10~5 copies/ng·DNA. Class I integron (intI1) has the highest content of 6.24×10-4 copies/ng·DNA in the pig house; the second is the chicken house 2.78×10-4 copies/ng·DNA; the lowest content of the cow house is 1.30×10-4 copies/ng ·DNA. Microbial aerosols in livestock and poultry breeding houses can be used as a repository for pathogens and resistance genes. There are significant differences in the concentration of pathogenic bacteria in the air and the pollution of resistance genes in different livestock and poultry houses. Therefore, it is of great significance to adopt different disease prevention and control methods and environmental control methods for different types of livestock and poultry breeding to reduce the pollution caused by the breeding environment to the surrounding environment.

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