【作者】 王伟；

【导师】 周立志；

【作者基本信息】 安徽大学 ， 生态学， 2021， 硕士

【摘要】 动物肠道微生物菌群的维持对宿主的生长发育和免疫稳态方面至关重要,跨物种传播影响肠道微生物菌群结构。在自然保护区内,候鸟的觅食栖息地点相对固定,周围存在许多养殖户散养的家禽活动。在一定区域内,远近不同的家禽群体可能直接和间接地对候鸟产生影响,并造成肠道微生物的交叉感染和跨种传播。因此,研究候鸟和家禽肠道细菌(包括潜在病原菌)的相互影响对加强野生动物栖息地管理具有现实和理论价值。本研究对越冬时期升金湖地区白头鹤(Grus monacha)及其1km内(D-N)和4km外(D-F)的家鸭(Anas platyrhynchos domesticus)进行粪便样品采集,并利用高通量测序技术(Illumina Hi Seq)和生物信息学分析软件分析白头鹤和家鸭肠道细菌及潜在病原菌的时空特征,主要结果如下:1.整个越冬期白头鹤和家鸭肠道菌群存在明显的跨种传播趋势,其中白头鹤与近距离家鸭在细菌OTU(Operational Taxonomic Unit)、优势菌门、核心菌方面存在更多的共享。白头鹤与家鸭肠道中有7944个细菌OTU,共有的细菌OTU占比为25.7%,其中白头鹤与远距离家鸭共享的OTU为20.2%。肠道中所有细菌包括四大优势菌门:厚壁菌门(Firmicute)、拟杆菌门(Bacteroidetes)、变形菌门(Proteobacteria)和蓝细菌门(Cyanobacyeria)。肠道核心菌门中,白头鹤和家鸭在蓝细菌门和放线菌门(Actinobacteria)差异显著(P<0.05),其中白头鹤肠道中差异菌群由门到种阶元共筛选出9个,近距离家鸭肠道中有34个,而远距离家鸭差异菌群只有2个。白头鹤和近距离家鸭肠道中共含有相对丰度最高的都是醋酸乳杆菌(Bacillus aceticus)和酸鱼乳杆菌(Lactobacillus acidipiscis),远距离的家鸭相对丰度最高的是乳酸杆菌属(Lactobacillus)。白头鹤和家鸭在肠道菌群群落组成上都存在差异,其中白头鹤和近距离家鸭肠道细菌群落存在更显著的聚集趋势(R=0.815,P=0.001)。2.越冬期变化下,白头鹤与家鸭肠道细菌交互传播现象逐渐明显,物种间距离越近,重叠程度越高,细菌群落组成差异越小。在越冬前、中、后时期的白头鹤肠道中共鉴定出24个门,其中各个越冬期重叠18个细菌门。近距离家鸭在越冬三个时期共鉴定出29个细菌门,各越冬期共享20个门。而远距离家鸭在整个越冬时期,共鉴定出27个肠道菌门,各个越冬时期共含有21个细菌门。随着越冬时期的变化,白头鹤的Alpha多样性差异由极显著(P<0.01)到不显著(P>0.05),近距离家鸭肠道的细菌OTU丰富度逐渐高于远距离家鸭。总体上白头鹤和家鸭肠道的差异菌群不断变化,在越冬中期最高达到45个差异菌群门类,白头鹤和家鸭群体肠道核心差异菌数量也不断改变,其贡献率也不断增加,其中变化最显著的有类芽孢杆菌属(Paenisporsarcina)、诺卡氏菌属(Nostocoid)、梭状芽孢杆菌属(Clostrium)等。3.白头鹤和家鸭肠道病原菌的感染程度与距离存在相关关系,在越冬时间变化下,感染程度逐渐加深,其中家鸭肠道中存在更多的潜在致病菌。白头鹤和家鸭肠道潜在致病序列占细菌总数的3.69%,家鸭群体致病序列数高于白头鹤。白头鹤潜在病原菌的Alpha多样性与家鸭群体差异显著(P<0.05),且近距离家鸭致病菌OTU丰富度高于白头鹤和远距离家鸭。肠球菌(Enterococcus cecorum)和肉毒梭菌(Clostridium botulinum)是经比对筛选出的最主要菌种,主要存在于白头鹤和家禽肠道中。随着越冬期变化,白头鹤和家鸭潜在致病菌序列的重叠度由越冬前期0.22%,增加到越冬后期的0.33%。本研究首次比较和分析,时空变化下白头鹤和不同远近距离家鸭肠道菌群的组成、共性和差异性,以及潜在病原体的多样性形成机制,为界定自然保护区野生鸟类与自由放养家禽的安全距离,探索珍稀鸟类传染病的传播机制提供了依据。更多还原

【Abstract】 The stable animal intestinal microflora plays a crucial role in growth,development of the host and immune homeostasis.The structure of intestinal microflora is affected by cross-species transmission.In the nature reserve,the foraging habitat of migratory birds is relatively fixed and domestic poultry is also present in the surrounding areas.Therefore,it is of practical and theoretical value to study the mutual influence of gut bacteria(including potential pathogenic bacteria)of migratory birds and domestic poultry for strengthening wildlife management.This study was conducted during over wintering period and fecal samples were collected from hooded cranes(Grus monacha)and two groups of domestic ducks(Anas platyrhynchos domesticus)present within 1 km(D-N)and 4 km away(D-F)in the Shengjin Lake area.By using high-throughput sequencing(llumina Hi Seq)technology and bioinformatics analysis software the temporal and spatial characteristics of intestinal bacteria and potential pathogens of hooded crane and domestic ducks were analyzed.The results of this study are as follows:1.An obvious cross-species transmission of intestinal microbes was observed between hooded cranes and domestic ducks throughout the wintering period.The hooded crane and domestic ducks present at distance of 1 km have more shared bacterial OTU(Operational Taxonomic Unit),dominant bacteria phyla,and core bacteria.A total of 7944 bacterial OTUs were present in the intestines of hooded cranes and domestic ducks,and the shared bacterial OTU accounts for 25.7%.The shared bacterial OTU between hooded crane and domestic ducks present at 4 km distance accounted for 20.2%.Four phyla were dominant across all samples i.e.Firmicute,Bacteroidetes,Proteobacteria and Cyanobacteria.The different intestinal bacterial groups were screened from phylum to specie level in this study.There were significant differences present among two core phyla cyanobacteria and Actinobacteria(P<0.05)in hooded cranes and domestic ducks.Thirty four bacterial groups were found in the intestines of domestic ducks present at near distance and two different bacterial groups were present in the intestines of domestic duck present at long distance.The two bacterial species,Bacillus aceticus and Lactobacillus acidipiscis with highest relative abundance were found in hooded cranes and domestic ducks present within close range.The intestinal bacterial community composition of hooded cranes and domestic ducks exhibited significant differences and have a more significant aggregation trend(R=0.815,P=0.001).2.During the changing winter period,the cross-transmission of intestinal bacteria between hooded cranes and domestic ducks become prominent.The smaller difference and significant overlap were observed in intestinal bacterial communities between species present at close distance.We identified a total of 24 bacterial phyla during three winter periods(early,middle and late)and 18 phyla overlapped in each period of winter.In domestic ducks a total of 29 bacterial phyla were found and among them 20 phyla were shared during three periods of winter.The domestic ducks present at a long distance(D-F)have a total of 27 bacterial phyla throughout winter period and there were 21 bacterial phyla in each wintering period.With the change of overwintering period,the alpha diversity of hooded cranes changed from extremely significant(P<0.01)to insignificant(P>0.05),and the intestinal bacterial OTU enrichment was gradually higher in domestic ducks at close distances than domestic ducks at long distances.In general,the differential flora of the intestines of hooded cranes and domestic ducks was constantly changing,reaching up to 45 differential flora categories in the middle period.The number of core bacteria in the intestinal tract of hooded cranes and domestic ducks showed a constant change,with increase in the contribution rate.The most significant changes are Paenisporsarcina,Nostocoid,and Clostrium.3.The cross transmission of intestinal bacterial pathogens of hooded cranes and domestic ducks is related to distance between the two species.The degree of cross-infection gradually increases with the changing winter period.The potential pathogenic sequences in the intestine of hooded cranes and domestic ducks accounted for 3.69% of the total number of bacteria,and the number of pathogenic sequences in domestic duck populations was higher than hooded cranes.The OTU richness of pathogenic bacteria was higher in domestic ducks than hooded cranes and the alpha diversity of potential pathogenic bacteria of hooded cranes showed significant difference than domestic ducks(P<0.05).Enterococcus cecorum and Clostridium botulinum were the most important species found in the intestine of hooded cranes and domestic ducks.The overlap of potential pathogenic bacteria sequences between hooded cranes and domestic ducks increased from 0.22% in the early overwintering period to0.33% in the late overwintering period.It is the first time to compare and analyze the composition,commonality and difference of the intestinal flora of hooded cranes and domestic ducks at different distances under temporal and spatial changes,as well as the formation mechanism of the diversity of potential pathogens in the study.This is the definition of wild birds and free-range poultry in nature reserves.The safe distance between the two species has provided a basis for exploring the transmission mechanism of rare bird infectious diseases.更多还原