The Application Of Metagenomic On Detection Of Pathogenic Microorganism And Analysis Of Microbial Community Profiles In Cattle’s Stomachs

This paper focus on the pathogenic microorganisms in the blood of the incidenceswine and cattle stomach microbial diversity using high-throughput Illuminasequencing platform and454sequencing platform.1. Applied research in the detection of pathogenic microorganismsmetagenomicsInfectious diseases outbrok in a pig farm in Chongqing and another pigfarm inHenan, with clinical symptoms were persistent fever, anemia, and pale mucousmembranes. Two pigs blood samples were collected from Chongqing farm and twopigs samples from Henan pigfarm, then blood metagenomes were extracted. Thesample1was sequencing repeatedly and marked the sample5.16S rDNA universalprimers of the V3V4region were used for PCR amplification, wherein the V3V4region fragments was obtained, which contained all the bacterias. Sequenceinformation of V3V4fragments was obtained by using454sequencing platform.Sample1obtained13,105reads, sample2obtained14,449reads, sample3obtained18,525reads, sample4obtained16,858reads, and sample5obtained15,202reads.After the primers and low quality sequences removed, the effective reads wereobtained, sample1obtained6,555effective reads, sample2obtained7,419effectivereads, sample3obtained10,824effective reads, sample4obtained9,270effectivereads, and sample5obtained7,747effective reads. By bioinformatic analysis,14phyla were detected, sequences of Proteobacteria, γ-Proteobacteria, Pasteurella,Pasteur Salmonella, and Haemophilus were the most amount in samples1,2and5,accounted for39.3%,35.6%and38.2%of the total, sequences of γ-Proteobacteria,Pasteurella, Pasteurella Branch, and Pasteur were the maximum amount in samples4and5, accounted for33.7%and35.9%of the total. The very little amount to fragments of other bacteria was detected, indicating that diseases were caused bypathogens in the two genera.The genomes were interrupted using ultrasound, and then adaptors wereconnected, the libraries were construct. Metagenomic fragments were sequenced byIllumina sequencing platform sample1was22,056,354reads, sample2was17,835,316reads, sample3was20,106,956reads, sample4was17,646,500reads.Afer low quality sequences and host sequences removed, the effective reads wereobtained, sample1obtained115,503effective reads, sample2obtained70,283effective reads, sample3obtained94,957effective reads, and sample4obtained67,359effective reads. By bioinformatic analysis,15kinds of pathogenicmicroorganisms and three viral sequences were detected in four samples.Haemophilusparasuis was the most abundant in sample1and sample2, accountingfor46.8%and43.3%of the total; Pasteurella multocida was the largest content insample3and sample4, accounting for42.5and45.9%of the total. Other fragmentsof bacteria were rarely detected, which consistent with the Illumina test results.In this paper, the results of the two pathogens were validated by PCR method,results of PCR experiments showed consistent with the high-throughput sequencingresults.2. Metagenomics analysis of the microflora composition of cow stomachThe samples of rumen, reticulum, omasum, and abomasum content were collectedfrom Changchun slaughter house. After pre-treatment, different stomach microbialmetagenomes were extracted, but for the technical reason, the metagenomic ofabomasum was not obtained. Universal primers of the16S rDNA V3region wereused for PCR amplification of the metagenomes. V3fragment sequence informationwas got by using Illumina sequencing platform, raw data was acquired, sample1was1.2Gb, sample2was1.2Gb, sample3was1.17Gb; Afer the primer and low qualitysequences removed, the effective reads were acquired, sample1was28.66Mb,sample2was31.04Mb, sample3was33.44Mb. The V3sequences were assigned to17phyla and most sequences were found to come from microorganisms of five phyla, i.e. Bacteroidetes(56%), Firmicutes(35%), Proteobacteria(5.5%), Spirochaetes(1.6%),and Lentisphaerae (1.1%). The three stomach compartments shared similar lineages ofbacteria phyla, and a total of13phyla were determined in the rumen,16in theomasum, and16in the reticulum. Further analysis of the sequences,68,83, and63genera WERE identified respectively in the three samples,46genera were detected inall the three samples. The top10most abundant genera of the three stomachs werecompared, and the significant difference was observed. The P values of all the top10most abundant genera were less than0.05(p<0.05).The study was the first application of metagenomics on the analysis and detectionof pathogenic microorganisms in swines blood, and revealed pathogenicmicroorganisms which might cause the diese, laying the foundation formetagenomics in the detection of pathogenic microorganisms in animals‘blood.Meanwhile this study was the first application using metagenomics approach inin-depth analysis of microflora in cattle reticulum and omasum, and revealedsimilarities and differences of cattle rumen, reticulum and omasum microflora. Thestudy was significant in learning physiological functions of rumen, reticulum andomasum, and the role of cow stomach microflora in health and disease.