| Dental caries is one of the most important diseases in captive giant panda(Ailuropoda melanoleuca) cubs,which has an important effect on their quality of life and future feeding ability.About 50% giant panda cubs may got dental caries during the childhood,which threating the healthy development of captive population.However,there still no studies focused on the development pattern,causing factors and carious mechanism of oral microbiota in captive giant panda cubs.Thus,our study collected oral samples from captive giant pandas aged 1 to 12 months in Chengdu Research Base of Giant Panda Breeding,and carried out the following three aspects of research:firstly,16S rRNA amplification sequencing was performed to monitoring the oral microbiota of giant panda cubs at different months of age,to explored the oral microbiota development patterns of panda cubs with dental caries and healthy ones;Secondly,mass spectrometry was used to analyze the factors affecting the occurrence of dental caries in giant panda cubs,and to analyzed the correlation with oral microbial diversity,microbial community composition and function.Thirdly,mass spectrometry,metagenomic and metabolomic was used to analyzed the differences and associations between the intake of different nutrients,oral microbiota and metabolites of 12-monthold panda cubs with or with not caries,and explored the differential metabolic pathways and the pathogenesis of caries.1.Study on the development of oral microbiota in captive giant panda cubsIn total of 90 saliva samples from 10 captive giant panda cubs (Caries Active group,CA,n=5;Caries Free group,CF,n=5) during 9 different months of age were analyzed for microbiota composition.Results:(1) The richness and diversity of oral microbiota of panda cubs in the CA and CF showed the same trend with the increase of development,but the change range of CF group was more tender.The richness of oral microbiota in the CA significantly decreased at 10 months of age (p=0.0079),and the diversity of oral microbiota significantly decreased at 5,8 and 10 months of age (p <0.05);(2) The oral micromiota structure of the two groups showed no difference in the first 3 months after birth,and showed significant difference from 4 to 12 months of age;(3) Actinobacillus was significantly enriched in CA from 4 to 12 months of age;(4)The types of functional metabolic pathways were the same between the two groups at 1 to 12 months of age,and the relative abundance differences were mainly concentrated in the 5,6 and 12 months of age.Carbohydrate metabolism,amino acid metabolism and other metabolic pathways were significantly enhanced in the CA at 12 months of age (p <0.05).The results showed that compared with CF individuals,the oral microbial community of panda cubs with dental caries was unstable and developed slowly,and the structure,composition and gene function of the oral microbial community began to show significant differences at 4 months of age.2.Study on carious factors of captive giant panda cubsIn order to exploring the carious factors,this part of study analyzed the 16S rRNA sequencing results of mother oral microbiota,phenotype data of cubs,behavioral development data,identification results of different nutrients in food by mass spectrometry and nutrient intake data at different stages of 1-12 months of age,and analyzed the correlation between the oral microbiota and KEGG level 2 metabolic pathways of giant panda cubs.Results:(1) Mother oral microbiota,birth weight,gender,twins,behavioral development time,breast milk and formula intake had no effect on caries of giant panda cubs.(2) Continuous excessive intake of sucrose,arabinose and various amino acids in the accumulated food intake of giant panda cubs resulted in decreased oral microbiota diversity and significantly increased Actinobacillus relative abundance,and significantly reduced in relative abundance of Lautropia,Rodentibacter,Streptococcus and Rothia,which promoted carbohydrate metabolism,amino acid metabolism and genetic information related metabolism pathway.Reduce secondary metabolite biosynthetic pathways.The results showed that continuous excessive intake of sucrose,arabinose and amino acids was an important factor leading to caries in captive giant panda cubs.3.Study on carious mechanism of captive young giant pandasTo investigating the mechanism of caries,the food components of 12 1-year-old captive giant panda cubs were identified by mass spectrometry,and their oral saliva samples were tested by metagenomic and metabolomic analysis,respectively.Results:(1) Among all 81 nutrients,the cumulative intake of 15 nutrients such as sucrose,histidine and glycine in CA was significantly higher than that in CF group.(2) Sucrose,histidine,glycine and other 12 nutrients significantly increased the gene abundance of bacteria in caries enrichment group,and increased the abundance of multiple genes involved in carbohydrate metabolism,amino acid metabolism,fatty acid metabolism and membrane transport metabolic pathways.(3) Genes related to carbohydrate metabolism,amino acid metabolism and membrane transport metabolic pathways were significantly up-regulated in the CA group,and were co-regulated by a variety of bacteria enriched in the CA group.(4) Among 775 different oral metabolites,391 were enriched in the CA group and 384 were enriched in the CF group,the metabolites in the CA group contained more organic acid compounds.(5) Bacteria and metabolic pathways in the caries group were significantly positively correlated with organic acid compounds in saliva.In conclusion,the high frequency supply of nutrients such as sucrose and amino acids in food increased the abundance of 9 kinds of bacteria(Brevibacterium linens),improved the metabolic pathway function of carbohydrates and amino acids,produces a large amount of butyric acid,causes oral environment acidification,eroded tooth enamel and leading to dental caries.Conclusion:(1) The oral microbiota of the captive giant panda cubs with caries and healthy had similar development trend from 1 to 12 months of age,but the development pattern was different.(2) Excessive sucrose and amino acids in food were important influencing factors of caries in captive giant panda cubs;(3) Sucrose and 12 amino acids could significantly promoted carbohydrate metabolism and amino acid metabolic pathway function of oral microbiota of captive giant panda cubs,leading to the accumulation of butyric acid and caries. |
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