| Bovine mastitis is the inflammation of mammary glands resulting from the infection of pathogenic microorganisms.It is prevalent in many dairy farms and brings about heavy economic losses,seriously endangering.the healthy development of dairy industry.Based on the clinical symptoms,bovine mastitis is usually divided into two types:clinical and subclinical mastitis,of which the latter with high incidence rate is easy to be neglected due to absence of any naked changes in both udder and milk and thus contributes to huger potential losses.At present,antibiotics remain the main drugs to treat bovine subclinical mastitis.However,with the constant emergence of drug-resistant strains and increasing attention on food safety problems,looking for a non-antibiotic method for treatment of bovine subclinical mastitis has been the research hotspot in recent years.Therefore,the present study focused on the therapeutic effect of Atractylodis macrocephalae Koidz.polysaccharide extracts(RAMP)with immunomodulatory activity on subclinically mastitic cows and its preliminary mechanism,and hoping to provide a novel method for clinical medication.1 Isolation,purification of RAMP and preparation of oil emulsionObjective:To isolate and purify RAMP,and process it into oil emulsion with stable physical behavior and suitable viscosity for injection.Methods:The crude RAMP was isolated by water extraction and ethanol precipitation,and then was subjected to deproteinization with Sevag method and dialysis.Subsequently,the total carbohydrate content in vacuum freeze-drying RAMP was measured by phenol-sulfuric method.The purified RAMP was processed.into oil emulsion(RAMP-O)under high-speed shearing with rapeseed oil and emulsifiers(tween 80 and span 80).Then the type identification,stability,viscosity,particle size and distribution of RAMP-O were analyzed.Results:RAMP separated and purified in the present study was non-starch and non-reducing polysaccharide and contained no proteins or nucleic acids.The yield and total sugar content of RAMP were 6.58%and 89.63%,respectively.The prepared RAMP-O was O/W ordinary emulsion with excellent stability and appropriate viscosity,and its Z-average particle size was 1130 nm.2 Therapeutic effect of RAMP-0 on bovine subclinical mastitisObjective:To investigate the therapeutic effect of RAMP-O on subclinical mastitis in cows.Methods:RAMP-0 was subcutaneously injected bilaterally in the area of supramammary lymph node(SMLN)in a healthy cow.Then the irritation of RAMP-0 was evaluted by closely observing the changes in the injection site and analyzing the composite milk SCC for three consecutive days after administration.The effects of RAMP-0 on quarter or composite milk SCC and NAGase activity were evaluated by injection of RAMP-O in the area of SMLN at affected side or at both sides in cows with SCC of more than 0.5 million cells/ml.No treatments were conducted in the control cows.Quarter or composite milk samples were collected before and 1,2,3 weeks after treatment for analysis of SCC and NAGase activity.The effects of RAMP-0 on milk SCC,NAGase activity,the number of infected quarters and milk quality were evaluated by injection of RAMP-O in the area of SMLN at both sides in subclinically mastitic cows.No treatments were conducted in the control cows.Quarter milk samples were collected before and 1,2,3 weeks after treatment for analysis of SCC,NAGase activity and bacteriology.Composite milk samples were collected 1 month after treatment for analysis of SCC and contents of protein and fat in a DHI center.The effect of RAMP-O on dairy herd milk SCC was evaluated by injection of RAMP-O in the area of SMLN at both sides in cows with SCC of more than 0.4 millon cells/ml.No treatments were conducted in the control cows.Composite milk samples were collected 1 month after treatment for analysis of SCC.Results:Subcutaneous injection of RAMP-0 in the area of SMLN had no visible side effects and significantly reduced quarter or composite milk SCC and NAGase activity via administration at affected side or both sides.Subcutaneous injection of RAMP-O in the area of SMLN at both sides significantly reduced composite and quarter milk SCC,quarter milk NAGase activity and the number of infected quarters,and had no side effects on daily milk yields and milk quality(milk fat and protein contents).Subcutaneous injection of RAMP-O in the area of SMLN at both sides reduced herd milk SCC in a large-scale dairy farm.3 Immunomodulatory effect of RAMP on bovine SMLN lymphocytesObjective:To investigate the immunomodulatory activity of RAMP in bovine SMLN lymphocytes in vitro.Methods:Crude RAMP was isolated by water extraction and ethanol precipitation and then was subjected to deproteinization by papain-Sevag combination method and dialysis to obtain the total RAMP(RAMPtp)and three fractional RAMPs(RAMP60,RAMP70 and RAMP80).FTIR and MTT methods were employed to compare their structural characteristics and stimulating activities in lymphocytes proliferation,respectively.RAMPtp was furtherly characterized by detections of molecular weight(Mw),monosaccharide compositions,chemical shifts and ultrastructure,and analyzed for its effects on intracellular Ca2+ concentration,cell cycle and cytokines(IFN-y,IL-4,IL-17A and TGF-?1)mRNA expression.In addition,fluorescein labelled RAMPtp(flRAMPtp)was prepared by cyanogen bromide(CNBr)-activated method and was employed to study the binding action between RAMPtp and lymphocytes.Results:The four polysaccharides showed different FTIR characteristics and only RAMPtp was found to promote lymphocytes proliferation among the three soluble polysaccharides(RAMP70,RAMP80 and RAMPtp).GPC showed that the number-average Mw of RAMPtp was 1867.HPLC indicated that RAMPtp was composed of glucose,mannose,rhamnose,arabinose and galactose with mass percentages of 60.67%,14.99%,10.61%,8.83%and 4.90%,respectively.NMR 1H and 13C spectra implied that RAMPtp was of both a-and?-configuration and mainly connected by 1,3-linked ?-D Galp and 1,6-linked ?-D Galp residues.SEM presented that RMPtp was amorphous in structure.RAMPtp could bind to receptors of lymphocytes,increase intracellular Ca2+ concentration,promote lymphocytes in G0/G1 phase into S and G2/M phases,upregulate IFN-y and IL-17A while downregulate IL-4 and TGF-?1 mRNA expression.4 Transcriptome analysis of bovine SMLN lymphocytes stimulated by RAMPtpObjective:To investigate the molecular mechanism involved in the effect of RAMPtp on bovine SMLN lymphocytes in vitro.Methods:Transcriptome analysis was conducted by RNA-Seq in lymphocytes stimulated by RAMPtp and bioinformatics analyses were performed based on differential expression,GO function and KEGG enrichment.Results:243 differential expression genes(DEGs)were identified in RAMPtp-stimulated lymphocytes,of which 189 genes were upregulated,whereas 54 genes were downregulated.GO enrichment analysis identified cell part,cellular process and binding as the top three GO terms.KEGG enrichment analysis identified 9 canonical pathways,of which more DEGs were identified in Cytokine-cytokine receptor interaction,Jak-STAT signaling pathway and Chemokine signaling pathway.In conclusion,the polysaccharide RAMP was extracted from Atractylodis macrocephalae Koidz.and processed into vegetable oil emulsion RAMP-O.Subcutaneous injection of RAMP-0 in the area of SMLN was found to decrease SCC and NAGase activity,reduce the number of infected quarters and to have no visible side effects on cows.The in vitro experiments showed that only RAMPtp had SMLN lymphocytes-stimulating activity among different fractional RAMP extracts.RAMPtp was furtherly subjected to structural characterization on the basis of GPC,HPLC,NMR and SEM,and was found able to bind to SMLN lymphocytes,increase intracellular Ca2+ concentration,promote lymphocytes into S and G2/M phases,upregulate IFN-y and IL-17A while downregulate IL-4 and TGF-?1 mRNA expression.243 DEGS,47 enriched GO terms and 9 significant canonical pathways were identified by RNA-seq.It was speculated that the therapeutic effect of RAMP-O on bovine subclinical mastitis might be associated with the immunomodulatory effect of RAMPtp on mammary glands. |
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