Profiling Of Intestinal Microbiota And Fecal Metabolome In Neonatal Diarrhea Calves

Calf diarrhea is a gastrointestinal disease syndrome with indigestion and diarrhea as clinical features in calves.It is one of the main causes of calf death before weaning and brings huge economic losses to the dairy and beef cattle breeding industry.Intestinal pathogen infection is the main cause of calf diarrhea.Studies have shown that Bovine Rotavirus(BRV),Bovine coronavirus(BCoV),K99 Escherichia coli and Cryptosporidium are responsible for calf diarrhea.The most important pathogen.In addition,Salmonella,Clostridium perfringens,Bovine Viral Diarrhea Virus(BVDV),Giardia,Bovine Norovirus(BNoV)and Nebovirus(Nebovirus,NeV),etc.It is also an important pathogen causing calf diarrhea.At present,due to the lack of effective vaccines and drugs,the problem of calf diarrhea has not been fundamentally solved.Recent studies have shown that intestinal flora plays an important role in intestinal health,and the occurrence of intestinal diseases is closely related to the disorder of intestinal flora.The composition and structure of the intestinal flora of calves with diarrhea will change significantly,but its change characteristics and the mechanism of action of the intestinal flora in the occurrence of calf diarrhea are still unclear:This study intends to conduct an epidemiological investigation of newborn calf diarrhea in some areas of Ningxia,conduct 16S rRNA amplicon sequencing analysis and metabolome analysis on diarrhea calf feces,screen indicator species and differential metabolites,and explore the intestinal tract of diarrhea calves The composition of the flora and the change characteristics of metabolites lay a theoretical foundation for further revealing the mechanism of intestinal flora in the occurrence of diarrhea in newborn calves.1.This study investigated the incidence of diarrhea in pre-weaning calves within 2 months of age on medium and large-scale dairy farms in some areas of Ningxia.The feces of healthy and diarrheal calves were tested for BRV,BCoV,BCoV,and diarrhea.Detection of pathogens such as BNoV,BVDV,NeV,K99 E.coli and Cryptosporidium,and the genetic evolution analysis of the main pathogens.The results showed that calf diarrhea in some areas of Ningxia in this study mainly occurred in calves within 30 days of age.BRV,BCoV,BNoV and Cryptosporidium may be the main pathogens causing diarrhea in calves before weaning,and they were detected in calves with diarrhea.The rates were 19.8%,9.43%,12.26%and 6.6%respectively,and the main form of infection was single infection.The results of genetic evolution analysis showed that the BRV and BCoV detected in this study were basically the same as those reported in China.BNoV was mainly GIII.2,and Cryptosporidium was mainly Cryptosporidium parvum.The above results indicate that BRV,BCoV,BNoV and Cryptosporidium are the main pathogens of newborn calf diarrhea in some areas of Ningxia,and BNo V has been widespread in neonatal calves in some areas of Ningxia.2.In this study,healthy and diarrhea calf feces were selected to analyze the diversity of intestinal flora.The results showed that the diversity and richness of the intestinal flora of calves with diarrhea were significantly reduced,and the composition of the flora was significantly different at the taxonomic level of phyla,class,order,family,and genus.The screening results of differential markers showed that Escherichia coli,Fusobacterium_mortiferium,Fusobacterium,Escherichia-Shigella,etc.were mainly significantly enriched in the feces of calves with diarrhea;Fusobacteria were significantly enriched in calves with BRV-related diarrhea Enrichment;Prevotella,Bacteroides_caecigallinarum,etc.are significantly enriched in BCoV-related diarrhea calves;Proteobacteria,Escherichia coli,Bacteroides fragilis,etc.are significantly enriched in BNoV-related diarrhea calves.The results of species network correlation analysis showed that the key flora in healthy calves are mainly Firmicutes bacteria,and the key flora in the intestinal flora of diarrhea calves are mainly Proteobacteria bacteria.3.In this study,LC-MS metabonomic analysis was performed on diarrhea and healthy calf feces.The results showed that compared with healthy calves,the fecal metabolic profile of diarrhea calves changed significantly.CKgroup and Dgroup screened 54 different metabolites,of which 51 metabolites such as lysophospholipids were significantly reduced(P<0.05).Three metabolites such as bacterosterone B were significantly increased(P<0.05);35 different metabolites screened in CKgroup and BRV group,of which 34 metabolites such as polyporostosterone B,PC(16:1(9Z)/16:1(9Z)),and desthiobiotin Substances are significantly reduced,and LysoPE(0:0/22:0)are significantly increased.The enrichment results of metabolic pathways showed that the differential metabolites of the CKgroup and Dgroup were mainly related to folate biosynthesis,biotin metabolism and glycerophospholipid metabolism pathways,and the differential metabolites of the CKgroup and BRV groups were mainly related to biotin metabolism.The results of correlation analysis between flora and metabolites showed that the above-mentioned differential metabolites were mainly significantly positively correlated with beneficial intestinal bacteria such as Faecalibacterium and Euryarchaeota,and significantly negatively correlated with Clostridium,Proteobacterium,and Escherichia.4.In this study,diarrhea and healthy calf feces were analyzed by targeted metabolome analysis of bile acids and short-chain fatty acids.The results showed that compared with healthy calves,the metabolic profiles of bile acids and short-chain fatty acids in the feces of diarrhea calves changed significantly.CKgroup and Dgroup group bile acid targeted metabolite analysis screened out four different metabolites:chenodeoxycholic acid,allocholic acid,Norcholic acid,and hyocholic acid.Among them,chenodeoxycholic acid and allocholic acid are among them.The feces of calves with diarrhea were significantly increased(P<0.05),and norcholic acid acid and hyocholic acid were significantly decreased(P<0.05);a total of 6 different metabolites were screened out by targeted metabolome analysis of shortchain fatty acids.Butanoic acid,acetic acid and hexanoic acid were significantly reduced in the feces of diarrhea calves,and isohexanoic acid,isobutyric acid and isovaleric acid were significantly increased.The results of metabolic pathway enrichment analysis showed that the differential metabolites screened in this study were mainly related to pyruvate metabolism.