【作者】 徐卫平；

【导师】 徐永平； Tim A.McAllister；

【作者基本信息】 大连理工大学 ， 生物化工， 2010， 博士

【摘要】 近十几年来,疯牛病、口蹄疫、高致病性禽流感等重大动物传染性疫病肆虐全球,给全世界动物疫病防治工作敲响了警钟。染疫动物肉尸及其相关副产物的无害化处理是动物防疫工作的重要组成部分,而传统的掩埋、焚烧、炼油等染疫动物尸体的处理方法,都不能达到安全、环保和可循环利用的目的。生物安全静态堆肥法作为一种安全、环保、经济实用的新兴染疫动物处理方法越来越受到人们的关注。但是,由于缺乏科学的评价体系,目前还很少采用该方法处理染疫动物尸体。因此,本课题以大型染疫动物牛尸为研究对象,考察生物安全静态堆肥法处理染疫动物尸体的效果；研究病原微生物的灭活规律和动物肉尸的降解规律；同时建立一种科学的评价体系,为无害化处理染疫动物尸体提供理论和实验依据。染疫动物生物安全静态堆肥的建立：2006年建造了第一个染疫动物生物安全静态堆肥(堆肥2006),建造过程是先用草垛围成长25m、宽5m、高2.4m、厚1.2 m的长方‘体形发酵仓,在整个发酵仓内铺放青贮饲料用塑料布以防止病原菌的扩散,接着铺放40cm厚的干草。干草上放置16头牛尸(平均重量为343kg),再覆盖160cm厚的牛粪,最后用塑料布覆盖封闭,静置发酵147天。期间通过热电偶检测堆肥80cm和160cm深度处的温度,并定期采集这两个深度处的堆肥样品,进行含水率的检测。结果表明,80cm深度处的温度升高较快,8天即可达到55℃以上,并保持在55～63℃的范围,持续时间长达35天以上；而160cm深度处的温度较低,但可保持40℃以上,持续4个月。检测堆中含水率结果可知,160cm深度的含水率显著高于80cm深度,147天时两个深度的含水率分别为74%和47%。导致该结果的主要原因,一是建造堆肥时使用的牛粪的含水率过高(68%),二是放置牛尸的位置太深(160cm)。牛粪中水的不断下沉和牛尸分解过程中释放的水分,都使160cm深度处的含水率增加,结果孔隙率降低,阻碍了空气的流通,进而抑制了微生物的活力,最终导致堆温的低下。因此,2007年建造了第二个生物安全静态堆肥系统(堆肥2007)。该堆肥是在2006年建造的基础上进行了两方面的改进：(1)降低牛粪的含水率,使用60%含水率的牛粪；(2)升高牛尸位置,在牛尸底部填入60cm厚的牛粪,使牛尸位于100cm深度,静置发酵230天。实验结果显示,这次(2007年)建造的生物安全静态堆肥在40cm,100cm和160cm深度处的温度,7天后均可达到55℃,而且持续70天以上。牛尸所处的100cm深度处的含水率在54～67%之间发生变化,适宜于微生物生长代谢的需求。因此,建造堆肥时牛粪中的含水率要控制在60%左右,牛尸要放在中间层深度,这样才能使堆温快速均衡的升高和长久保持。病原微生物的灭活规律：以大肠杆菌O157:H7 (Escherichia coli 0157:H7)、空肠弯曲菌(Campylobacter jejuni)和新城疫病毒(Newcastle disease virus)作为模式病原微生物,分别以109CFU／g,108CFU／g,106EID50／g的浓度接种于牛粪中考察其在堆肥80cm和160cm深度处的灭活规律。检测结果表明,大肠杆菌0157：H7和新城疫病毒第7天就被全部灭活,而空肠弯曲菌仍有活性。其主要原因是空肠弯曲菌为嗜热菌,具有耐热性。另外,将大肠杆菌O157：H7以109CFU／g的浓度分别接种于密封的无菌牛粪中和新鲜牛粪内,密封的无菌牛粪中病原菌仅受到堆肥热压力的作用,于28天才被全部灭活,而新鲜牛粪中的病原菌受到温度、pH和微生物的抑生作用等因素的影响,于7天内全部灭活,说明堆肥发酵产生的高温是杀死病害微生物的主要因素之一,而较高的pH值(9左右)与优势微生物的抑菌作用也有一定的杀灭病原微生物的作用。在生物封闭堆制结束时(堆肥2006第147天；堆肥2007第230天),上、中、下三层深度的大肠菌数量都小于1.0 log10 CFU／g dry wt,达到了较理想的灭菌状态,完全符合美国USEPA(2003)和加拿大CCME(2005)制定的大肠菌数量小于3.0 log10 CFU/g dry wt的目标要求。建立检测牛源DNA方法：选用Real-time PCR的SYBR Green法对牛源DNA进行检测和分析。以牛线粒体DNA序列为靶点,设计灵敏度高、特异性强的一对引物,扩增171bp的牛线粒体DNA片段(Mt171)。堆肥样品首先经过冷冻干燥后粉碎成粒径小于0.25μgm的粉末,再用DNA提取试剂盒提取总DNA,然后通过Real-time PCR进行检测和分析。由于DNA提取液中含有腐植酸等杂质可抑制PCR反应,因此通过优化确立了PCR体系中添加牛血清白蛋白的反应体系。实验结果表明添加牛血清白蛋白不仅可提高Real-time PCR效率,还可以减化DNA的纯化步骤。分析检测牛源DNA.运用优化的Real-time PCR方法检测牛线粒体DNA的结果表明,堆肥2006中牛源Mt171基因片段在80cm深度的降解速度明显快于160cm深度。80cm深度处的牛源Mt171片段含量自第7天开始逐渐减少,147天时的含量比第0天减少了79%；而160cm深度处的Mtl71片段的含量虽然也在减少,但147天时比第0天只减少了20%。而堆肥2007的100cm深度处Mt171片段含量的降解速度大幅提高,与第0天相比第112天的牛源Mt171片段含量减少了75%,第230天减少了86%。说明堆肥2007牛源Mt171片段的降解效率明显高于堆肥2006。上述实验结果表明,可以通过Real-time PCR方法检测牛源特异DNA片段的含量来衡量堆肥的发酵效率。牛尸组织的降解规律：堆肥中牛尸组织器官的降解速度依次为牛脑>牛蹄>牛骨。牛脑在堆制7天后的干重减少量>90%,牛蹄堆制56天后的干重减少量>80%,而牛骨在堆制147天后的干重减少量只有15%。检测牛脑中牛源DNA片段Mt760(牛线粒体基因片段长度为760bp)和Mtl71(牛线粒体基因片段长度为171bp)的降解速度结果显示,7天时Mt760和Mt171片段的减少量分别达到91%和84%；230天时的减少量分别达100%和99%。而检测牛脑附近牛粪中的Mt760和Mt171片段的数量在第7-28天显著增加,第28～230天缓慢下降,第230天时比第0天分别减少了94%和75%。说明牛尸组织器官的降解是牛尸体内牛源细胞被不断降解,并逐渐释放到牛体外,再通过周围环境(牛粪)中微生物的作用进一步降解的过程。本研究所建生物安全静态动物堆肥系统,7天后上、中、下三个深度的温度均可达到55℃以上,且可持续70天之久,适于处理感染了由温度敏感的病原微生物引起的染疫动物尸体。所建生物安全静态动物堆肥法不仅满足美国USEPA(2003)和加拿大CCME(2005)关于静态堆肥规定的温度要达到55℃、持续3天以上的要求,而且高温分布的均匀性和持续性也都超出了已报到的相关动物堆肥系统。生物安全静态堆肥法,作为一种安全、环保、高效、可循环利用的方法,将为处理染疫动物尸体提供新的手段,也为相关行业标准的确立提供了实验和理论依据。更多还原

【Abstract】 Recent high-profile outbreaks of infectious animal diseases (e.g., mad cow disease, foot and mouth disease, and avian influenza) emphasize the need for safe methods of mortality disposal. Comparing to the conventional methods of burial, incineration and rendering, biosecure mortality composting could be a reliable and practical alternative for disposal of infectious carcasses and manure. However, the utilization of animal carcass composting was limited, which was partially caused by the lack of understanding of pathogen inactivation profile and animal tissue degradation efficiency. The present study aimed to develop a biosecure static composting system for disposal of cattle carcasses and manure in the event of an infectious disease outbreak, investigate the pathogen inactivation rate and cattle tissue degradation efficiency, and finally construct the theoretical and experimental foundation for the using of biosecure static composting system as a carcass biosafe disposal method in the event of animal disease outbreak.To develop a biosecure cattle mortality composting system, we construct duplicate biosecure structures in 2006 (Compost2006), containing 16 cattle (Bos taurus) mortalities (343 kg average weight) with carcasses placed on a 40 cm straw layer and overlaid with 160 cm of feedlot manure. The whole compost material was covered by the silage plastic all over the sides and built in a barley straw bale rectangle bin of 25 m length,5 m width,2.4 m height, and 1.2 m thickness. Temperature was monitored daily by the thermal couples and moisture content was determined at intervals at 80 and 160 cm depths (P80, P160) over 147 d composting period. At P80, compost heated rapidly, exceeding 55℃after 8 d and maintained temperatures of 55-63℃for>35 d. At P160, temperature failed to exceed 55℃, but remained above 40℃for>4 mo. Using manure of 68% initial moisture content could be one of the reasons that P160 failed to reach 55℃, as the downward pooling of water resulted in over-wet condition of 79% moisture content at P160 after 147 d and prohibited the oxygen transfer and the microbial activity. Although the cattle were deeply degraded after 147 d, raising carcasses higher and using manure with lower moisture content may improve heating in the area surrounding carcasses and enhanced the bovine tissue degradation. Thus, we built duplicate compost structures again in 2007 (Compost2007) with the modification of (1) carcasses were raised to 100 cm depth by placing them on an additional 60 cm layer of manure and (2) feedlot manure with 60% moisture content at construction was used. The following temperature profiles at depths of 40,100, and 160 cm (L40, L100, L160) all reached 55℃after 7 d, and maintained above 55℃for at least 70 d over 230 d biosecure composting. The moisture content at carcass depth varied between 54-67%, suitable to the microbial growing. Thus, placing carcass at middle depth and using manure of approximately 60% moisture content play important roles in the efficient compost heating.To investigate rates of microbial inactivation in Compost2006, Escherichia coli O157:H7, Campylobacter jejuni, and Newcastle disease virus (NDV) were inoculated in manure (E. coli O157:H7 and C. jejuni≈108 CFU/g; NDV,≈106 EID50/g), embedded at P80 and P160 and retrieved at intervals during composting. E. coli O157:H7 and NDV were undetectable after 7 d at both depths. The C. jejuni DNA was detected up to 84 d at P80 and >147 d at P160. Compost heat was the primary cause of pathogen inactivation, while factors other than heat also contributed. At the completion of biosecure composting (d 147 of 2006, and d 230 of 2007), levels of total coliforms were all below 1.0 log10 CFU/g dry wt at top, middle and bottom layers of structures, which sufficiently meet the requirements of USEPA (2003) and CCME (2005) as less than 3.0 log10 CFU/g dry wt.Real-time PCR SYBR Green Assay was developed to determine the concentration of bovine specific DNA in compost samples. A pair of specific and sensitive primers targeting 171 bp bovine mitochondrial (Mt) DNA fragment (Mt171) was designed. Compost DNA was extracted from freeze-dried and ground samples using DNA extraction kit. To eliminate the inhibition effects caused by the coextracted humid substances, the PCR system was optimized by adding bovine serum albumin (BSA) and diluting the DNA solution. The modification improved the real-time PCR efficiency and avoided the DNA purification process.Bovine DNA degradation in Compost 2006 and 2007 was studied using the optimized real-time PCR method. In Compost2006, bovine specific DNA degraded faster at P80 than P160. Compared to d 0, copies of the Mt171 fragment were reduced by 79% at P80, but only 20% at P160 after 147 d. In Compost2007, copies of the Mt171 fragment were degraded 75% by d 112 and 86% by d 230 at L100. Thus, the modification at construction in Compost2007 extremely improved the bovine DNA degradation at the carcass depth compared to Compost2006. Quantification of bovine specific DNA using real-time PCR assay could be an efficient method to evaluate the degradation efficiency of material of bovine origin.Bovine tissue decomposition ranked as brain>hoof>bone. More than 90% dry matter (DM) of brain disappeared after 7 d and 80% DM of hoof decomposed after 56 d, while only 15% DM of bone degraded after 147 d. The bovine Mt760 (a 760 bp bovine Mt-DNA fragment) and Mt171 fragments decomposed rapidly in bovine brain tissue (BT), and were degraded by 91%,84% by d 7, and 100%,99% by d 230, respectively. However, in the manure surrounded brain tissue (BM), copies of Mt760 and Mt171 increased substantially over d 7-28 but decreased gradually thereafter and reduced compared to d 0 by 94% and 75% after 230 d, respectively. The carcass degradation process was thus characterised by the primary cell decomposition within the decayed carcasses and the further infusion into the surrounding manure following the secondary deeper decomposition by the compost heterotrophic microbes.The present study developed and optimized a biosecure composting system degraded 16 mature cattle carcasses per structure in a period of 5-7 mo. The key points of high temperature composting include placing carcasses at middle layer surrounding with sufficient cattle manure, using feedlot manure of optimal moisture content (60%) and C/N ratio (>15), insulating compost matrix from cool environment by straw bales and retaining moisture content with plastic sheeting. As the biosecure composting system used inexpensive materials and was relatively simple to construct, it shows promise for use in disease outbreaks where emergency on-farm disposal of carcasses is required. The biosecure system could be sized to dispose of thousands of carcasses provided that sufficient space and stockpiles of manure were available.The rapid suppression of the model pathogens E. coli O157:H7, coliforms and NDV indicates the biosecure composting system can dispose of infectious carcasses and manure contaminated with similar bacteria or viruses that are sensitive to heat and the chemical properties of compost.更多还原