Comparison And Analysis Of Intestinal, Oral And Genital Microorganisms Among Common Laboratory Animals And Human

Research BackgroundMicroorganisms are a type of tiny creatures, and very difficult to be observed by the naked eyes. They are a major component of life on Earth, playing a very important role in promoting material circulation, regulating climate balance and breaking down pollutants. Microorganisms play a key role in sustaining human health and the environment. They affect human health in many aspects such as growth, development, digestion, nutrient absorption, energy supply, fat metabolism, immune regulation, drug metabolism and so on. Microbes are widely distributed in the human body surface, mOTUh, respiratory tract, nose, intestines, vagina, urogenital tract and other parts, which we called the flora. Human beings are such a "super organism" composed of 10% human cells and to 90% of microbial cells. The human body has abOTU 1.5kg habitats microorganisms, which commensurate with the weight of the body’s liver. Total number of bacteria has reached 1014, which is abOTU 10 times as the human cells, and their genome is 1,000 times as much as the human genome. Microbes in the human body can be divided into two categories, settled flora and temporary flora. There are three relationships between the host and the flora including mutualists, commensals and pathogens. Settled bacteria and host benefit each other and interdependent. On the one hand, the host provides nutrition and living space for bacteria to settle in; on the other hand, settled bacteria help the host fending off the invasion of pathogens, in order to maintain a healthy body. Settled communities have a significant impact on human development, immune function, nutrient absorption and energy metabolism. Savage, a famous American professor of microbiology named the micro-ecological system as the tenth largest human respiratory system, such as circulatory, digestive, etc. In the medical field, human settlement commensal flora is defined as one of the "the other genome ". In recent years, our recognation of the human body structure and composition of flora is significantly improved by applying the 16S rRNA gene sequencing methods.Rats, rabbits, dogs, pigs and monkeys are experimental animals commonly used in biomedical research. With the demanding for more and more experimental animals, the studies of microbial communities in experimental animals gradually developed. Especially in recent years, sequencing of bacterial 16S rDNA-based second-generation high-throughput sequencing technology, with a high throughput,and low-cost advantages, can significantly improve the shortcomings of traditional microbiological research methods, and obtain ideal data of microbial communities. It has laid a good foundation for our research methodology microbial ecology, so that we can better recognize them.Research ObjectivesBy comparison microflora, microbial community structure and their respective characteristics and differences of several common laboratory animals were studied, such as Tibet mini-pigs, beagle dogs, macaques, New Zealand rabbits and Wistar rats, along with comparative analysis of normal human beings.The purpose of this research was to hold preliminary discussions on differences and similarities between microflora of animal models commonly used and human beings in order to provide basic data for the micro-ecological aspects of human medical research, and to provide several suggestions for choosing animal models used in the research on microecosystem.Research MethodsChapter IFresh manure, which was acquired from Tibet mini-pigs (adult) of the Songshan Lake Pearl Laboratory Animal Science and Technology Co., Ltd. in Dongguan, and beagles (adult), macaques (adult), New Zealand rabbits (adult), Wistar rats (adult) of SOTUhern Medical University Experimental Animal Center, was collected in EP tube. 20 samples that were composed of four ones each species, including one male and three female,were preserved at-20 ℃. The data of human intestinal flora was provided by the Institute of Public Health and Tropical, SOTUhern Medicine University.After extracting genomic DNA of the stool sample,16s RDNA bacterial universal primers, PCR amplified samples conserved region of 16S rRNA V4. High-throughput sequence data under the original machine was collected after PE100-bp sequencing of PCR end product via Illumina Hiseq 2000, preliminary processed of the original sequence in the BIPES (Barcoded Illumina Paired-end (PE) Sequencing) procedure. Clean sequences were clustered into OTUs(Operational Taxonomic Units), three distance methods during the process of which were used: The farrest, the nearest and the average distance method. OTUs obtained were classified using GAST (Global Alignment for Sequence Taxonomic) algorithm. αdiversity analysis(Shannon index, PD_whole_tree, Observed OTU, Chaol.ACE) of samples,that was numbers of species in a single sample,was acquired by the mothur method. βdiversity analysis,that was comparison of microbial community structure between different samples, was conducted by principal component analysis using QIIME. Linear discriminant analysis Effect Size (LEfSe) was conducted using linear discriminant analysis. Flora markers (that is Biomarker) of significant differences between different groups were searched using Galaxy89 online tool. LEfSe analysis was conducted firstly by Kruskal-Wallis rank and test to check species that the abundance difference between the inspection team was significant, then by group Wilcoxon rank and test to check species that has significant differences and finally by LDA to conduct data dimension reduction and evaluate impact(that is LDA score) of species that had significant differences. Statistical analysis was conducted using IBM SPSS Statistics 20 software.Pairwise comparative analysis based on normally distributed data was conducted using One-Way ANOVA in order to calibrate test level.The construction of PD_whole_tree、Shannon etc. was conducted by GraphPad prism5.Chapter ⅡFresh samples of oral flora, which were acquired from Tibet mini-pigs (adult) of the Songshan Lake Pearl Laboratory Animal Science and Technology Co., Ltd. in Dongguan, and beagles (adult), macaques (adult), New Zealand rabbits (adult), Wistar rats (adult) of SOTUhern Medical University Experimental Animal Center, was collected using disposable cotton swab.20 samples that were composed of four ones each species, including one male and three female,were preserved at -20℃. The data of human oral flora was provided by the Institute of Public Health and Tropical, SOTUhern Medicine University. After extracting genomic DNA of the stool sample, 16s RDNA bacterial universal primers, PCR amplified samples conserved region of 16S rRNA V4. High-throughput sequence data under the original machine was collected after PE100-bp sequencing of PCR end product via Illumina Hiseq 2000.Methods of processing data were similar with Chapter One.Chapter ⅢFlora samples of reproductive tract, which were acquired from Tibet mini-pigs (adult) of the Songshan Lake Pearl Laboratory Animal Science and Technology Co., Ltd. in Dongguan, and beagles (adult) and macaques (adult) of SOTUhern Medical University Experimental Animal Center, was collected using disposable cotton swab. Samples were composed of three ones each species and were preserved at -20℃.The data of human reproductive tract flora was provided by the Institute of Public Health and Tropical, SOTUhern Medicine University. After extracting genomic DNA of the stool sample,16s RDNA bacterial universal primers, PCR amplified samples conserved region of 16S rRNA V4. High-throughput sequence data under the original machine was collected after PE100-bp sequencing of PCR end product via Illumina Hiseq 2000.Methods of processing data were similar with Chapter One.ResultsChapter I1、In a diversity analysis, there were significant difference between people, pigs and dogs in terms of PD_whole_tree index, meanwhile, pigs and macaques, dogs, rabbits, rats compared remarkable difference(p<0.003), the remaining species had no significant difference with each other(p>0.003); As for Shannon index, there were significant difference between people and the pig gut microbiota, Tibet mini-pigs and beagle, New Zealand rabbits compared remarkable difference(p<0.003), the remaining species had no significant difference with each other(p>0.003).2、βdiversity showed that Tibet mini-pigs and macaques had the most relevant similarity.3、 In microbial community structure level, Intestinal flora gate structures of the Tibet mini-pigs and human were most similar; Dogs’gate structure level was similar to rats. Human’s gut flora Bacteroides accounted for the largest proportion in flora structure genera, Prevotella, Faecalibacterium, Roseburia and Fusobacterium toke the ratio with human intestinal flora more than three-fifths. Prevotella accounted for a large proportion in intestinal flora of pigs, macaques and rats, but intestinal flora of pigs mainly consisted of Prevotella, Fusobacterium and Treponema; Lactobacillus and Oscillibacter were the two main part in intestinal flora of macaques; intestinal flora of rats dominated by Fusobacterium and Variovorax. Sphingomonas, Fusobacterium and Lactobacillus were the main component of intestinal flora of dogs; intestinal flora of rabbits dominated by Lactobacillus,Bacteroides and Ruminococcus.Chapter Ⅱ1.20 samples of Tibet mini-pigs, Beagles, macaques, New Zealand rabbits, Wistar rats and 15 samples of human oral, after quality control we acquired 939,272 clean 16S rRNA gene sequences, and the minimum was 101, all samples were included in the subsequent analysis.2. a diversity analysis showed the oral flora of Shannon index of people, Tibet mini-pigs,beagle dogs, macaques, New Zealand rabbits and Wistar rats were not significantly different (p> 0.05). Due to PD_whole_tree index did not follow a normal distribution, Taking advantage of non-parametric method of analysis, people and pigs, dogs, macaques, rabbits, had a significant difference (p<0.05), pigs and dogs, macaques, rabbits compared remarkable difference (p<0.05), the remaining species had no significant difference with each other (p> 0.05).3. Through the analysis of βdiversity of Tibet mini-pigs, beagle dogs, macaques, New Zealand rabbits, oral flora richness of Wistar rats relatively similar, which caused by experimental animals lived in a specific environment. In relative terms, macaques, pigs and people live in a closer distance, therefore, macaques and pigs showed similarity with human oral flora colonies than other species. After weighted, taking into account the abundance relationship, macaques and people had a closer distance and higher similarity.4. There were five doors bacteria predominate in oral flora gate structure level, respectively were:Actinobacteria, Firmicutes, Proteobacteria, Bacteroides and fusobacteria. While oral flora gate structure levels of pigs, dogs, macaques, rabbits, rats, were all Actinobaceria and Firmicutes with more than fifty percents. gate structure level of pigs, macaques, rats were broadly similar, actinomycetes, Bacteroides and Firmicutes were all existed in their body. In comparison, the level of oral flora in pigs were more closer to people. In the case of oral bacteria level there were a large difference, but in general, Neisseria, Roseburia, porphyromonas, Moraxella, oral streptococci, actinomycetes, Fusobacterium, Lactobacillus were all existed in those five species.Chapter Ⅲ1.9 samples from Tibet mini-pigs, beagles and macaque, plus 10 samples from healthy people aged 30 to 50, totally 19 samples, were quality controlled, and then we acquired 135,191 clean 16S rRNA gene sequences. The obtained lowest 1935 gene sequences and 19 samples were all included in the subsequent analysis.2. a diversity differences had no statistically meaning, indicating pigs, dogs, macaques and human reproductive tract flora may not have significant differences in the type and quantity.3. β diversity analysis with the same species unweighted, human and macaque reproductive tract flora was closer; while with weighted, human and miniature reproductive tract was quite similar.4. Human, pigs, dogs and macaques reproductive tract phylum level were mainly Firmicutes, Bacteroidetes, Proteobacteria and Actinobacteria. Moreover, the reproductive tract flora phylum levels of Tibet mini-pigs and human were quite similar, Firmicutes, Bacteroidetes, Proteobacteria, Actinobacteria accounted for all the flora phylum more than 90% level, and phylum structure level of dogs and macaques were similar, Fusobacteria accounted for more than 10 to 20 percents. Genus level distributions of four species were greatly different, even if the different samples from the same species were quite different. But on the whole, the Prevotella, Lactobacillus, Streptococcus and fusobacterium, which were contained in human reproductive tract, also appeared in pigs, dogs and macaques reproductive tract flora.Conclusions1. The intestinal flora structure of the Tibet mini-pigs, Beagles, macaques, New Zealand rabbits, Wistar rats and human are different. Comparatively speaking, the intestinal flora phylum structure level of the Tibet mini-pigs and human is the closest, and the dogs and rats are relatively similar.2. The oral flora structures of Tibet mini-pigs, Beagles, macaques, New Zealand rabbits, Wistar rats and human are different. At the level of genus, Fusobacterium is one of the observed indicators of the treatment effect of periodontal disease. The results also show that oral flora Clostridia is slightly larger than other animals. In addition, porphyromonas is one of the oral floras that are related to periodontal disease, whose proportions of dogs and macaques are closer to human.3. At the level of phylum, the reproductive flora of human, Tibet mini-pigs, beagle dogs and macaques are mainly Firmicutes, Bacteroidetes, Proteobacteria, and Actinobacteria, among which pigs have the closer phylum structure level to human.