Comparative Study On Mammary Microflora Of Dorper Sheep And Small Tailed Han Sheep By High-throughput Sequencing

Microbial community compositions and diversity of mammary gland are closely related to mammary gland health and disease status.However,the sources and influencing factors of mammary gland microorganisms remain elusive to date.This study was aimed to elucidate the effects of intrinsic genetic factors including sheep breed and extrinsic environmental factors including feed and soil on the compositions and relative abundance of mammary gland bacterial communities.In this study,16 Dorper(DM)and 17 Small Tailed Han(SM),4 feed(F),2 soil(S)inside and outside the enclosure were selected as the research subjects.33 sheep milk samples(1 milk sample per sheep),4 feed and 2 soil samples were collected.High-throughput sequencing of bacterial 16 S r DNA gene with RDP and SILVA database was used to analyze the structural compositions and relative abundance of bacterial communities.Excel 2016 was used to analyze basic data and draw histogram figures,Vegan package of R language was used to draw cluster heat-maps,Canoco 5.0 software was used for principal component analysis(PCA).Meanwhile,California Mastitis Test(CMT)was used to identify positive milk samples with mastitis from 14 Dorper(DM)and 30 Small Tailed Han(SM)sheep without clinical mastitis.Furthermore,the positive detection rate was investigated based on the presence of three main pathogens of mastitis(Staphylococcus aureus,Escherichia coli and Streptococcus agalactiae)by PCR techniques.A total of 125 the bacterial taxa at family level were obtained across 39 milk-feed-soil samples.Based on relative abundance of the top 10 dominant families each sample,a total of 43 bacterial families were obtained for subsequent analysis.The results revealed that Bacillaceae and Staphylococcaceae were the most dominant taxa in the mammary bacterial communities of Dorper(DM)and Small Tailed Han(SM)sheep with the highest relative abundances of 98.35%(DM3)and 94.95%(SM3),respectively.Bacillaceae and Staphylococcaceae(>20.00% relative abundance)respectively accounted for 37.50%(6/16)and 18.75%(3/16)of Dorper(DM)sheep mammary bacterial communities.Bacillaceae and Staphylococcaceae(>20.00% relative abundance)respectively accounted for 58.82%(10/17)and 29.40%(5/17)of Small Tailed Han(SM)sheep mammary bacterial communities.Nocardiaceae was the sub-dominant taxa in the mammary bacterial communities of Dorper(DM)and Small Tailed Han(SM)sheep,the maximum relative abundance was 21.46%(DM9),whose proportion of mammary bacterial communities(relative abundance >5.00%)was 43.80%(7/16)and 17.60%(3/17)in Dorper(DM)and Small Tailed Han(SM)sheep,respectively.Pseudomonadaceae ? Leuconostocaceae ?Enterobacteriaceae were the most dominant families in the feed bacterial communities,with maximum relative abundances in F1(59.49 %),F1(33.80%),and F3(35.05%),respectively.Ruminococcaceae ?Rikenellaceae ?Planococcaceae were the most dominant families in the soil bacterial communities,with the highest relative abundances in S2(29.40%),S2(14.24%)and S1(13.28%),respectively.Cluster heat-map analysis showed that the majority of the Dorper and Small Tailed Han sheep mammary bacterial families were clustered into a large group with high similarity to the soil(S)bacterial families,while that the minority of the Dorper(DM)and Small Tailed Han(SM)sheep mammary bacterial families were clustered into a large group with high similarity to the feed(F)bacterial families.Principal component analysis(PCA)showed that the mammary bacterial communities of Dorper(DM)sheep was highly correlated with the majority of mammary bacterial communities,while the Bacillaceae and Staphylococcaceae were highly correlated with the mammary bacterial communities of Small Tailed Han(SM)sheep.In summary,the compositions and relative abundance of mammary bacterial communities were closely related to sheep breed,which were also influenced by feed and soil environmental microbiota factors.CMT-based result showed that 2 weakly positive samples(DM2,DM10)and 3 weakly positive samples(SM8,SM9,SM26)from 14 Dorper(DM)and 30 Small Tailed Han(SM)sheep without clinical mastitis,respectively.PCR result showed that Staphylococcus aureus,Escherichia coli and Streptococcus agalactiae were not detected in the 5 weakly positive mastitis samples from 2 Dorper(DM2,DM10)and 3 Small Tailed Han(SM8,SM9,SM26)sheep.In conclusion,these results underpin tracing contagious pathogens(e.g.,S.aureus and S.agalactiae)and environmental pathogens(e.g.,E.coli)leading to mammary mastitis as well as prevention and control of mastitis from different breeds of sheep.