| Diarrhea is a common disease in the dairy farm,which has extremely high incidence in unweaned calves,seriously threatens the health of calves,and may negatively affect their subsequent production performance.The gut microbial disturbance is an important cause of diarrhea in calves.With the rise of high-throughput sequencing and multi-omics technologies,the role of gut microbiota in calf diarrhea has been deepened.However,due to complex factors such as feeding management and environment,the gut microbiota structure of calves in different dairy farms may change and the pathogens that cause diarrhea may be special.Knowing the relationship between the calf gut microbial structure and diarrhea in specific large-scale dairy farm can better guide the prevention and treatment of diarrhea.Therefore,the purpose of this study is to analyze the effect of the fecal microbiota of calves on diarrhea,and provide experimental basis for the follow-up prevention and treatment of calf diarrhea in this dairy farm.48 female calves were selected in this experiment,and their daily diarrhea scores were recorded from birth to one week after weaning.Blood and rectal fecal samples were collected to detect relevant physiological and biochemical indicators,and 16 S rDNA sequencing technology was used to analyze the structure of fecal microbiota of calves.Finally,association analysis and model prediction were used to explore the role of fecal microbiota in calf diarrhea.The test results are as follows:(1)The overall diarrhea rate was 6.40 %,and the diarrhea rate between the age of replacing milk replacer and the age of 45 d was the highest,which was 10.68 %.During diarrhea,the fecal microbiota of calves changed greatly.The alpha diversity indices(Ace,Chao1 and Observed species)in 35-day-old healthy calves were significantly lower than those in 35-day-old diarrhea calves(P < 0.01).In terms of relative abundance,Bifidobacterium,Peptostreptococcus,Streptococcus and Eubacterium＿hallii＿group in 15-day-old diarrhea calves were significantly lower compared to 15-day-old healthy calves(P < 0.05).The unidentified＿Oscillospiraceae,Allisonella,Sellimonas,Prevotella＿9 and Prevotellaceae＿UCG-001 in 35-day-old diarrhea calves were significantly lower compared to35-day-old healthy calves(P < 0.05).In absolute number,Bifidobacterium in 15-day-old diarrhea calves was significantly lower compared to 15-day-old healthy calves(P < 0.05).Total bacterial count,Faecalibacterium,Prevotella,unidentified＿Oscillospiraceae and so on in 35-day-old diarrhea calves was significantly lower compared to 35-day-old healthy calves(P < 0.05).(2)Compared to the same age,the absolute concentration of butyrate in feces of 15-day-old diarrhea calves significantly decreased(P < 0.05).The proportion of acetate of 15-day-old diarrhea calves significantly increased,while the proportion of butyrate in feces significantly decreased in both 15-day-old diarrhea calves and 35-day-old diarrhea calves(P< 0.05).Total antioxidants,malondialdehyde,globulin and IL-6 in serum and SIgA in feces of 35 d calves showed no significant difference between diarrhea calves and healthy calves(P > 0.05),but the content of zonulin in the feces of 35 d calves significantly increased in diarrhea calves(P < 0.05).Antibiotic resistance genes Fex A,BlaTEM,BlaCTX and BlaSHV were prevalent in calf feces.The relative abundances of BlaTEM and BlaCTX were affected by calf age and diarrhea status.The relative abundance of BlaTEM in 35-day-old diarrhea calves was higher than that in 15-day-old diarrhea calves(P < 0.05).The relative abundance of BlaCTX in 35-day-old diarrhea calves was significantly higher than that in 35-day-old healthy calves and 15-day-old diarrhea calves(P < 0.01).(3)The relative abundances of Megamonas(P < 0.05,R =-0.37),unidentified＿Oscillospiraceae(P < 0.01,R =-0.42)and Phascolarctobacterium(P < 0.01,R =-0.50)were significantly negatively correlated with the proportion of acetate in feces,and the relative abundances of Faecalibacterium(P < 0.05,R = 0.35)was significantly positively correlated with the proportion of butyrate in feces.The relative abundances of Alloprevotella(P < 0.01,R =-0.68),Prevotella(P < 0.01,R =-0.59)and Sellimonas(P < 0.05,R =-0.49)were significantly negatively correlated with zonulin in feces.The relative abundances of Allisonella was significantly negatively correlated with Ace(P < 0.01,R =-0.59),Chao1(P < 0.01,R =-0.60)and Observed species(P < 0.01,R =-0.56)indices,while the relative abundances of Romboutsia was significantly positively correlated with Ace(P < 0.05,R =0.44),Chao1(P < 0.01,R = 0.44)and Observed species(P < 0.01,R = 0.45)indices.The relative abundances of Megamonas(P < 0.05,R = 0.49),Prevotella(P < 0.01,R = 0.60)and so on were significantly positively correlated with fecal total bacterial count,and the relative abundances of Clostridium＿sensu＿stricto＿1(P < 0.01,R =-0.57)was significantly negatively correlated with fecal total bacterial count.The relative abundances of Lactobacillus was significantly negatively correlated with the relative abundance of fecal BlaCTX(P < 0.01,R =-0.33)and Fex A(P < 0.05,R =-0.28),while The relative abundances of Escherichia Shigella was significantly positively correlated with the relative abundance of fecal BlaSHV(P < 0.05,R = 0.26).In the microbial interaction network of healthy calves,the nodes such as Peptoclostridium,Erysipelotrichaceae＿UCG-003,Alloprevotella,Blautia,Ruminococcus＿torques＿group are many connected and located in the center of the network.In diarrhoeal calves,Escherichia Shigella had increased participation in the entire network,while other probiotics Bifidobacterium,lactobacillus,Limosilactobacillus,Akkermansia and so on had decreased or disappeared participation.Through the regression prediction of the calf health score in the random forest model,the predicted TOP25 genus were clustered into two categories and negatively correlated with each other.Among them,Prevotella＿9 and some other genera were significantly positively correlated with calf health score,and Clostridium＿sensu＿stricto＿1 was significantly negatively correlated with calf health score.In summary,diarrhea is common in calves on this farm.During diarrhea,the abundance of probiotics in calves decreased and the abundance of pathogenic bacteria increased.The fecal microbiota of calves changes in structure and function greatly,and it has significant correlation with fecal VFA,zonulin,antibiotic resistance genes and so on.Some bacterial genera play an important role in the microbial interaction network and have a good predictive effect on diarrhea,which are the key microorganisms associated with calf diarrhea. |
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