Study On The Changes Of Gastrointestinal Microbiota Of Piglets By Using 16S RRNA Gene-based Molecular Methods

The mammalian gastrointestinal(GI) tract habors a dense and complex microbiota, which plays an important role in nutrition,immunity and physiology of host.More attention should be paid to the study on the GI microbiota of piglet since it will suffer the special physiological phase like early-weanlng.This present study was carried out to investigate the changes of microbioia in GI tract of piglets from 7 to 35(two weeks after weaning) days of age by using 16S ribosomal RNA(rRNA) gene based molecular methods. Changes in bacterial community composition in stomach,jejunum and ileum of piglets after weaning was investigated using denaturing gradient gel electrophoresis(DGGE),and real-time PCR assay was also used to quantitatively analyze changes in predominant bacterial community.Bacterial and Lactobacillus community between digestia and mucosa samples in jejunum and ileum of piglets were also compared in this thesis.Representational difference analysis(RDA) was used to compare two L.sobrius strains isolated from piglets in different area.16S rRNA-based methods were also used to monitor changes in the hindgut bacterial community of piglets after oral administration of L.sobrius S1.Finally,to explain the limitation of 16S rRNA gene-based methods,sequences alignment analysis was performed to compare 16S rRNA genes of different Lactobacillus spp.based on different variable region fragments.1 PCR-DGGE analysis of stomach total bacterial and lactobacilli community in sucking and weaned pigletsMolecular methods based on the 16S rRNA gene were used to study bacterial and lactobacilli communities in stomach of neonatal piglets from 7 to 35(two weeks after weaning) days of age.The results showed stomach pH value was about 3.0 before weaning, while the pH value increased apparently after weaning.The semi-quantitative dilution-PCR showed that bacterial concentration in stomach of piglet was very low before weaning, while increased significantly after weaning.Compared with total bacteria,lactobacilli concentration increased slightly.The DGGE profiles from total bacteria in stomach showed the bands of samples before weaning were very simple and the predominant band belonged to far-migrating bacterium.After weaning,some new predominant bands appeared and the previous far-migrating bands disappeared simultaneously.The similarity of DGGE profiles from stomach lactobacilli showed weaning stress had no obvious effect on lactobacilli community.2 Changes of microbiota in stomach,jejunum and ileum of piglets after weaningChanges of bacterial community composition in stomach,jejunum and ileum of piglets after weaning(21 day) were investigated by 16S ribosomal RNA(rRNA) gene-based molecular methods.Dehaturing gradient gel electrophoresis(DGGE) profiles from bacterial community in foregut of piglets showed that,after weaning,predominant bands related to Lactobacillus spp.disappeared and substituted by some potential pathogenic species such as Peptostreptococcus anaerobius,Moraxella cuniculi,S.suis and Porphyromonas catoniae. Real-time PCR assay revealed that the percentages of lactobacilli and Lactobacillus sobrius relative to total bacteria were significantly lower in foregut samples of weaned piglets compared with the 21-day piglets.Here,we also described S.suis-specific real-time PCR assay,which can quantify S.suis in complex gastrointestinal microbiota with the 16S rRNA gene concentration higher than 10 copies per microliter DNA sample.This assay showed that S.suis predominated in stomach samples of weaned piglets with population levels up to 10~7 cells per gram of digesta,although this species was not detected in stomach of pre-weaning piglets.3 Comparison of bacterial and Lactobacillus community between digestia and mucosa samples in jejunum and ileum of pigletsTo investigate the differences of bacterial and Lactobacillus community between digesta and mucosa samples in jejunum and ileum of piglets from 7 to 35 days of age,using 16S rRNA gene based methods.DGGE profiles from bacterial community showed that the L.sobrius-related band was also observed in jejunal mucosa of piglets,while the predominant band related to S.suis observed only for weaned piglets did not appear in profiles generated from mucosa samples.Primers(Bact-0027/Lab-0677, Lab-0159/Uiv-0515) were used to amplify the Lactobacillus-specific 16S rRNA gene fragments from digesta and mucosa samples,the PCR products were then analyzed by DGGE.Specific bands were observed in DGGE profiles derived from mucosa-associated bacteria.However,16S rRNA gene sequence analysis indicated that the bands were most closely related to alfa-proteobacteria(Brevundimonas vesicularis).Further sequence alignment revealed that there were one or two bases mismatches between Lactobacillus-specific primers(Lab-0159) and target sequences of 16S rRNA gene from the bands related to alfa-proteobacteria.4 Comparison of two Lactobacillus strains isolated from piglets in different area by using representational difference analysisRepresentational difference analysis(RDA) has been adapted recently to study the genomic diversity of bacterial strains.Two Lactobacillus strains(L.sobrius 001~T and Lactobacillus sp.S1) were isolated from the intestine of piglets in difference area.Highly similarity of 16S rRNA gene between the two strains was found by DGGE and sequencing analysis.Both of these two strains could be detected in 1.2%agarose gel by Lactobacillus sobrius-specific PCR assay.RDA showed that genomic DNA of the two strains was digested with Cfoâ… ,similar profiles of DNA fragments were observed between the strains. After subtractive hybridization,no RDA amplified products were found,which suggested that there was no genomic difference between these two strains.The results indicated that strain S1 also belonged to L.sobrius,strain 001~T and S1 were the similar strain.5 16S ribosomal RNA-based methods to monitor changes in the hindgut bacterial community of piglets after oral administration of L. sobrius S1Changes in the composition of microbiota in the hindgut of piglets were studied after oral administration of L.sobrius S1,using molecular techniques based on 16S rRNA genes. Six litters of neonatal piglets were randomly divided into control and treatment groups.At 7, 9 and 11 days of age,piglets in the treatment group orally received a preparation of strain S1.V6-V8 and V2-V3 variable regions of the total bacterial and Lactobacillus-specific 16S rRNA gene pool,respectively,were amplified by PCR and analyzed by denaturing gradient gel electrophoresis.DGGE analysis showed that several populations present in the hindgut of piglets,represented by far-migrating bands,disappeared after weaning.Sequence analysis indicated that most of these bands corresponded to Lactobacillus spp.This trend could also be confirmed by plate counting and quantitative real-time PCR.Drastic changes of L.amylovorus and L.sobrius in total lactobacilli populations were also observed in the colon of piglets around weaning,as monitored by Lactobacillus-specific PCR-DGGE. Comparison of DGGE profiles between control and treatment groups revealed a specific band related to Clostridium disporicum that was found in treatment group on day 14.On day 35,a specific band appeared only in the control group,representing a population most closely related to S.suis(99%).Species-specific real-time PCR revealed that the population of L.sobrius which has the same position as strain S1 in DGGE profiles declined apparently in the colon of piglets after weaning,while it tended to come back earlier after oral administration of strain S1.These results indicated that specific populations(mainly Lactobacillus spp.) disappeared from the predominant bacterial community in the porcine hindgnt after weaning.Oral administration of strain S1 could mitigate this disruption, thereby contributing to the prevention of pathogen overgrowth during the critical phase of weaning.6 Sequences alignment of Lactobacillus spp.16S ribosomal RNA genes based on different variable region fragmentsPCR- DGGE based on 16S rRNA gene is commonly used to examine bacterial communities in a variety of environments.However,some unexpected results were observed in recent studies.For example,a single band can be identified to several bacterial species,and several different bands identified to the identical species.Here,we aligned the 16S rRNA gene sequences based on different variable region fragments using Lactobacillus spp.as reference strains.The results showed some different Lactobacillus species possessed the identical partial 16S rRNA gene sequences.Based on different variable region fragments,remarkable difference was observed on pairwise distance in the reference 16S rRNA gene sequences.0.061 of divergence value was found in V6-V8 region of 16S rRNA gene sequences between two L.arnylovorus clones,although they had the identical V2-V3 variable regions.These results indicated that the limitation of DGGE was to some extent due to the characteristic of bacterial 16S rRNA gene sequences.The results also indicated that more attention should be paid to the sequence difference of PCR products among different species,as well as to the specificity of primers when designing new primers for DGGE-PCR.