改性蒙脱石作为抗生素对断奶仔猪肠道菌群的影响

Animal Feed Science and Technology 198(2014)257–262Contents lists available at ScienceDirect

Animal Feed Science and

Technology journal homepage:

3592f506168884868662d60e/locate/anifeedsci Effects of cetylpyridinium-montmorillonite,as alternative to

antibiotic,on the growth performance,intestinal micro?ora

and mucosal architecture of weaned pigs

Y.L.Ke,L.F.Jiao,Z.H.Song,K.Xiao,3592f506168884868662d60ei,J.J.Lu ?,C.H.Hu ??

Animal Science College,Zhejiang University;The Key Laboratory of Molecular Animal Nutrition,Ministry of Education,

Hangzhou 310058,China a r t i c l e i n f o Article history:

Received 7July 2014

Received in revised form 8October 2014

Accepted 9October 2014Keywords:

Antibiotic

Cetylpyridinium-montmorillonite

Intestinal micro?ora

Mucosal architecture

Weaned pigs a b s t r a c t Cetylpyridinium-montmorillonite (CP-Mt)was used as an alternative to chlortetracycline on growth performance,intestinal micro?ora and morphology in weaned pigs.A total of 150piglets (Duroc ×Landrace ×Yorkshire,weaned at 21±1d age)were allotted to ?ve groups:(1)control;(2)control +0.5g/kg CP-Mt;(3)control +1.0g/kg CP-Mt;(4)control +1.5g/kg CP-Mt;(5)control +75mg/kg chlortetracycline.Each treatment has six pens with ?ve pigs per pen.The feeding trial lasted three weeks.The results showed that incremental CP-Mt inclusion in the diet increased average daily gain (linear P=0.013;quadratic P=0.034),decreased Escherichia coli and Streptococcus suis in jejunal contents linearly (P=0.004and P=0.006)and quadratically (P=0.014and P=0.009),decreased E.coli and Streptococcus suis in colon linearly (P=0.005and P=0.001)and quadratically (P=0.007and P=0.004),increased

villus height (linear P<0.001;quadratic P=0.001)and the ratio of villus height and crypt

depth (linear P<0.001;quadratic P=0.001),reduced plasma diamine oxidase (DAO)(linear

P=0.000;quadratic P=0.001),and increased jejunal mucosa DAO (linear P=0.006;quadratic

P=0.002).The piglets fed 1.0g/kg and 1.5g/kg CP-Mt did not differ from those fed chlorte-

tracycline (P>0.05)in growth performance,intestinal micro?ora and mucosal architecture.

The results indicated that CP-Mt could be a good candidate as an antibiotic alternative in

weaned pigs.

?2014Elsevier B.V.All rights reserved.1.Introduction

Early-weaned piglets commonly encounter low feed intake,body weight loss and post-weaning diarrhea (Hu et al.,2013b;McLamb et al.,2013;Smith et al.,2010).There is a large number of evidence showing that weaning disturbs intestinal microbiota and impairs intestinal morphology (Hu et al.,2012b,2013c;Jiao et al.,2014).Antibiotic is widely used to reduce enteric infections and the occurrence of pathogens able to adhere to intestinal mucosa (Kil and Stein,2010).The increased use of antibiotics has given rise to a concern of the development of resistant pathogenic bacterial strains and residual Abbreviations:ADFI,average daily feed intake;ADG,average daily gain;SEM,standard error of the mean;CP-Mt,cetylpyridinium-montmorillonite;DAO,diamine oxidase.

?Corresponding author.??Corresponding author.Tel.:+8657186961553;fax:+8657186961553.

E-mail addresses:jjlu@3592f506168884868662d60e (J.J.Lu),chhu@3592f506168884868662d60e (C.H.Hu).

3592f506168884868662d60e/10.1016/j.anifeedsci.2014.10.010

0377-8401/?2014Elsevier B.V.All rights reserved.

258Y.L.Ke et al./Animal Feed Science and Technology198(2014)257–262

contamination of the food chain with antibiotic(Vondruskova et al.,2010).In2006,the use of antibiotics as growth promoters was forbidden in the EU.

Special attention has been paid to natural clay minerals,which were not only used as antibiotic alternatives(Philip,2013; Song et al.,2012),but also used as carriers of antibacterial agent(Hu and Xia,2006;?zdemir et al.,2010).Montmorillonite (MMT),an aluminosilicate clay,has been generally used as an antimicrobial carrier due to its large speci?c surface area and high cation exchange capacity.The recent studies are mainly focused on immobilization of inorganic antimicrobial cations such as Cu2+and ZnO onto montmorillonite(Hu et al.,2012a,2013a;Song et al.,2013b).

Immobilization of organic cations with antibacterial activity has some advantages compared to that of inorganic cations (Herrera et al.,2000).Cetylpyridinium chloride as a quaternary ammonium salt is such an agent used in antiseptic solutions, and?nds a wide range of antibacterial applications in oral rinses and throat lozenges,in the meat industry for the decon-tamination of carcasses,and in the further processing of poultry,pork,and beef for the control of microbial growth(?zdemir et al.,2013).Previous investigations demonstrated that cetylpyridinium-montmorillonite(CP-Mt)exhibited strong antibac-terial activity for pathogenic bacteria,such as Salmonella enteritidis and Staphylococcus aureus(Herrera et al.,2000;?zdemir et al.,2013).

However,there are no data on the effects of CP-Mt in vivo.In this experiment,we hypothesized that CP-Mt could be used as alternative to chlortetracycline on growth performance,intestinal micro?ora and mucosal architecture in weaned pigs.

2.Materials and methods

2.1.Materials

The montmorillonite(Chifeng WHTB Mining Co.,LTD,Chifeng,China)content was99.0%.The cation exchange capacity (CEC)was1.30moL(+)/kg montmorillonite.The cetylpyridinium-montmorillonite(CP-Mt)was synthesized according to the method of Herrera et al.(2000)and?zdemir et al.(2013).Montmorillonite was hydrated with water to which cetylpyridinium chloride was added at an amount of0.5times the CEC of the montmorillonite.The resulting mixture was then agitated and allowed to exchange for24h.The CP-Mt was then separated by centrifugation and washed under agitation for24h with 100mL deionized water.The washed material was dried at60?C for24h and ground.

2.2.Experimental design and samples collection

All procedures were approved by the University of Zhejiang Institutional Animal Care and Use Committee.A total of150 weaning pigs(Duroc×Landrace×Yorkshire),with an average initial weight of6.2kg weaned at21±1d,were allocated to the?ve groups for three weeks,each of which was replicated six times with?ve pigs per replicate.The?ve groups were: (1)control;(2)control+0.5g/kg CP-Mt;(3)control+1.0g/kg CP-Mt;(4)control+1.5g/kg CP-Mt;(5)control+75mg/kg chlortetracycline.Diets were formulated to meet or exceed requirements suggested by the National Research Council(1998) (Table1).All pigs were given ad libitum access to feed and water.Average daily gain(ADG),average daily feed intake(ADFI), and gain/feed ratio were measured.

After the feeding trial,six piglets from each treatment(one pig per pen)were killed based on average body weight. Blood samples were collected from the anterior vena cava into tubes containing sodium heparin and mixed immediately to avoid coagulation.Plasma was obtained after centrifugation at3000×g for15min at4?C and then stored at?80?C until analysis.Specimens of the distal jejunum and proximal colon were?xed in10%formalin for morphology measurements. The intestinal contents from distal jejunum and proximal colon were collected for microbiota analysis.Mucosal samples from the distal jejunum were collected,rapidly frozen in liquid nitrogen and stored at?80?C for further analysis.

2.3.Sample analysis

16S ribosomal RNA-based methods were used for the abundances of Escherichia coli and Streptococcus suis as described by Huijsdens et al.(2002)and Su et al.(2008).Total DNA was extracted from intestinal contents using a TIANamp Stool DNA Kit(Tiangen Biotech),(TaKaRa Biotechnology,Dalian,China)according to the manufacturer’s instructions.Real-time PCR was performed on7500real time PCR systems(Applied Biosystems,Foster City,USA)using Fast SYBR?Green Master Mix SYBR Green chemistry(Applied Biosystems,Foster City,USA).For E.coli the forward primer sequence was 5 -CATGCCGCGTGTATGAAGAA-3 ,the reverse primer sequence was5 -CGGGTAACGTCAATGAGCAAA-3 (Huijsdens et al., 2002).For Streptococcus suis the forward primer sequence was5 -CAGTATTTACCGCATGGTAGATAT-3 ,the reverse primer sequence was5 -GTAAGATACCGTCAAGTGAGAA-3 (Marois et al.,2004).

Three cross-sections for each jejunal sample were stained with hematoxylin and eosin using standard paraf?n embedding procedures.Crypt depth and villus height were measured in at least10well-oriented crypt-villus units using image analysis (Leica Imaging Systems Ltd.,Cambridge,England)and averaged for each sample.

Diamine oxidase(DAO;EC1.4.3.6)activities in plasma and intestinal mucosa were measured using an enzymatic spec-trophotometric assay as described by Hu et al.(2012a).DAO activities in plasma and intestinal mucosa were expressed as U/mL and U/mg protein,respectively.

Y.L.Ke et al./Animal Feed Science and Technology198(2014)257–262259

Table1

Ingredient and chemical composition of the basal diet as fed basis.

Ingredients(g/kg)

Corn560

Soybean meal160

Extruded full-fat soybean120

Fish meal39

Spray-dried plasma protein25

Dried whey50

Soybean oil15

Dicalcium phosphate11.5

Limestone5

Sodium chloride3

l-Lysine HCl1

dl-Methionine0.5

Vitamin-mineral premix a10

Analyzed composition(g/kg)

Digestible energy b(MJ/kg)14.35

Crude protein224.5

Lysine14.6

Methionine 4.3

Calcium9.4

Total phosphorus7.5

a Provided per kilogram of diet:vitamin A,5500IU;vitamin D3,500IU;vitamin E,40IU;ribo?avin,5.0mg;vitamin B12,0.03mg;pyridoxine,3.0mg; vitamin K3,1.0mg;biotin,0.10mg;thiamine,2.0mg;niacin,30mg;pantothenic acid(calcium pantothenate),20mg;folic acid,0.6mg;choline(choline chloride),800mg;Zn(ZnSO4),100mg;Fe(FeSO4),125mg;Cu(CuSO4·5H2O),16mg;Mn(MnSO4·H2O),15mg;I(KI),0.2mg;Se(Na2SeO3),0.3mg.

b Digestible energy was calculated from data provided by Feed Database in China(2012).

2.4.Statistical analysis

Statistical analysis was performed by the SPSS9.0statistical package(SPSS Inc.,Chicago,IL).All treatments were compared by ANOVA using Tukey’s correction for multiple mean comparison.Polynomial contrasts were also used to assess the linear and quadratic effects of dietary CP-Mt inclusion level.Alpha level for determination of signi?cance was0.05.

3.Results

3.1.Growth performance

Growth performance of piglets is presented in Table2.Incremental CP-Mt inclusion in the diet increased ADG(linear P=0.013;quadratic P=0.034).As compared with control,supplementation with chlortetracycline improved(P<0.05)ADG. The growth performance of fed with CP-Mt did not differ from those fed with chlortetracycline(P>0.05).

3.2.Intestinal microbiota

Intestinal micro?ora of pigs is presented in Table3.Incremental CP-Mt inclusion in the diet decreased E.coli and Strepto-coccus suis in jejunal contents linearly(P=0.004and P=0.006)and quadratically(P=0.014and P=0.009).Incremental CP-Mt inclusion in the diet decreased E.coli and Streptococcus suis in colon linearly(P=0.005and P=0.001)and quadratically (P=0.007and P=0.004).The abundances of E.coli and Streptococcus suis of pigs fed CP-Mt diet did not differ from those fed chlortetracycline(P>0.05).

Table2

Effect of cetylpyridinium-montmorillonite on growth performance.

Control CP-Mt a(g/kg)Chlortetracycline SEM b P c

00.5 1.0 1.5Linear Quadratic ADG d(g)266y275xy286x285x290x 5.90.0130.034

ADFI e(g)3633623703693727.40.3750.681

Gain/feed0.730.760.770.770.780.020.1590.292

a CP-Mt,cetylpyridinium-montmorillonite.

b Standard error of means.

c Effect of CP-Mt addition by polynomial contrasts.

d ADG,averag

e daily gain.

e ADFI,average daily feed intake.

xy Means within a row with different letters differ signi?cantly(P<0.05).

260Y.L.Ke et al./Animal Feed Science and Technology198(2014)257–262

Table3

Effect of cetylpyridinium-montmorillonite on intestinal microbiota a and mucosal architecture.

Control CP-Mt b(g/kg)Chlortetracycline SEM c P d

00.5 1.0 1.5Linear Quadratic

Jejunum E.coli7.20x 6.83xy 6.51y 6.42y 6.30y0.180.0040.014 Jejunum Streptococcus suis 6.20x 5.58xy 5.22y 5.26y 5.19y0.230.0060.009 Colon E.coli8.42x7.93xy7.65y7.69y7.62y0.170.0050.007 Colon Streptococcus suis7.67x7.10xy 6.73y 6.57y 6.48y0.230.0010.004

Villus height(?m)629y680y767x763x758x25.5<0.0010.001

Crypt depth(?m)35033832032332912.60.0820.196

Villus height:crypt depth 1.80y 2.01y 2.40x 2.37x 2.30x0.070.0010.001 Plasma DAO(U/mL) 2.55x 2.13xy 1.79y 1.63y 1.73y0.16<0.0010.001 Jejunal mucosa DAO(U/mg protein)0.19y0.24xy0.28x0.26x0.26x0.020.0060.002

xy Means within a row with different letters differ signi?cantly(P<0.05).

a Bacterial numbers are expressed as log10(16S rRNA gene copies g?1wet weight).

b CP-Mt,cetylpyridinium-montmorillonite.

c Standar

d error of th

e mean,n=6.

d Effect of CP-Mt addition by polynomial contrasts.

3.3.Intestinal morphology

Table3shows jejunal morphology of pigs.Incremental CP-Mt inclusion in the diet increased villus height(linear P<0.001; quadratic P=0.001),and the ratio of villus height and crypt depth(linear P=0.001;quadratic P=0.001).The piglets fed1.0and 1.5g/kg CP-Mt did not differ in jejunal morphology from those fed Chlortetracycline(P>0.05).

3.4.Plasma and intestinal mucosa DAO

Plasma and jejunal mucosa DAO activity is presented in Table3.Incremental CP-Mt inclusion in the diet reduced plasma DAO(linear P<0.001;quadratic P=0.001),and increased jejunal mucosa DAO(linear P=0.006;quadratic P=0.002).The DAO activity of pigs fed CP-Mt diet did not differ from those fed chlortetracycline diet(P>0.05).

4.Discussion

In recent years,clay mineral-based antibacterial complexes have been prepared by a series of processes between the clay minerals and antibacterial substances(Hu and Xia,2006;?zdemir et al.,2010).Montmorillonites are a subset of alu-minosilicate clays having a2:1layer structure.Within the layers of these clays,substitution of other metal ions for silicon or aluminum can occur resulting in a net negative charge on the surface of the clay platelet.This negative charge is off-set by hydrated cations,the predominant ones being Na+and Ca2+(Herrera et al.,2000).These interlaminar cations can be exchanged with antibacterial cations,such as Cu2+and Zn2+(Malachova et al.,2011).The copper-exchanged montmorillonite exhibited strong antibacterial activity(Hu and Xia,2006).In vivo studies showed that copper-exchanged montmorillonite and ZnO-montmorillonite hybrid alleviated diarrhea(Hu et al.,2012b),modi?ed intestinal micro?ora and mucosal barrier integrity of weaned pigs(Hu et al.,2012a,2013a;Song et al.,2013b).However,there is increasing evidence that antimicrobial resistance genes are positively correlated with Cu and zinc concentrations in the environment(Choudhury and Srivastava, 2001;Mark et al.,2000).Many studies showed that heavy metals including copper and zinc,used in animal farming might promote the spread of antibiotic resistance via co-selection and/or other mechanisms(Baker-Austin et al.,2006;Holzel et al., 2012).Lina et al.(2011)found that zinc resistance and the czrC gene are widespread among CC398MRSA isolates.This sug-gested that the use of zinc in feed might have contributed to the emergence of antimicrobial resistance.Thus Cu-exchanged montmorillonite and ZnO–montmorillonite hybrid might also contribute to antimicrobial resistance.

Clay-based organic anti-bacterial agents were prepared by a reaction between clay and organic cations with antibacterial activity,and have some advantages compared to that of inorganic cations(Herrera et al.,2000),such as increased strength and heat resistance and increased biodegradability(Sinha Ray and Okamoto,2003)and longer duration of antibacterial ef?cacy(?zdemir et al.,2013).Cetylpyridinium chloride(CP)is a member of a family of quaternary ammonium salts which include a positively charged hydrophilic ammonium moiety and hydrophobic alkyl chains.The antimicrobial activity of CP is due to its ability to alter the permeability of cellular membranes allowing intracellular ions and low molecular-weight metabolites to diffuse out(Herrera et al.,2000).Depletion of these intracellular constituents causes the bacterial cell to consume available energy stores in an attempt to restore homeostasis,resulting in cellular death(Herrera et al.,2000). Previous investigations demonstrated that cetylpyridinium-montmorillonite(CP-Mt)exhibited strong antibacterial activity for pathogenic bacteria in vitro,such as Salmonella enteritidis and Staphylococcus aureus(Herrera et al.,2000;?zdemir et al., 2013).The present study showed for the?rst time that supplementation with CP-Mt decreased E.coli and Streptococcus suis in intestinal contents.Exchanging montmorillonite with CP produces a material that has a net positive charge and is more hydrophobic than the parent clay(Herrera et al.,2000;?zdemir et al.,2013).The CP-exchanged clay enhanced hydrophobicity and af?nity for pathogenic bacteria.Two possible mechanisms for the antibacterial activity were proposed.

Y.L.Ke et al./Animal Feed Science and Technology198(2014)257–262261 One model involved the adsorption of the bacteria and immobilization on the surface of the organoclay.Alternatively,CP could disassociate from the clay surface and directly exerts its antimicrobial effect on the bacteria(Herrera et al.,2000;?zdemir et al.,2013).

The structure of the intestinal mucosa can reveal some information on gut health.Stressors can lead relatively quickly to changes in the intestinal mucosa(Song et al.,2013a).Weaning is associated with villus atrophy and crypt hyperplasia(Hu et al.,2013b;Wijtten et al.,2011).A shortening of the villus decreases the surface area for nutrient absorption.The crypt is the area where stem cells pide to permit the renewal of the villus,and a large crypt indicates fast tissue turnover and a high demand for new tissue.In the present study,increase in villus height and villus height to crypt depth ratio at the small intestinal mucosa of the pigs supplemented with CP-Mt was observed.Such improved intestinal mucosal morphology may be related to the changes in intestinal microbiota,and in the present study numbers of E.coli and Streptococcus suis lowered.

The integrated intestinal barrier is to prevent the translocation of intestinal bacteria,and the entering of toxic or allergenic substances from the gut into the body(Wijtten et al.,2011).Injured intestinal barrier increased the epithelial permeability (Wijtten et al.,2011).The intestinal mucosa DAO activity is closely related with villi maturation and protein synthesis of mucosal cells.The normal healthy mucosa prevents the DAO in the intestinal lumen from entering the circulation.The DAO is normally present in very small amounts in the circulation(Wolvekamp and de Bruin,1994).When the intestinal barrier function is compromised,increased mucosal permeability allows more DAO to enter the peripheral circulation.Plasma DAO has been proposed as circulating markers for monitoring the extent of intestinal barrier injury(Fukudome et al.,2014).A large body of evidence has demonstrated that the early weaning process impaired intestinal barrier function of piglets(Hu et al.,2013b;McLamb et al.,2013;Smith et al.,2010).In the present study showed that CP-Mt supplementation increased intestinal mucosa DAO while reduced plasma DAO,indicating the barrier function were improved by CP-Mt feeding.

5.Conclusion

Supplementation with1.0g/kg and1.5g/kg CP-Mt in weaned diets was as effective as chlortetracycline for improving growth performance,mucosal architecture and modifying intestinal micro?ora.The CP-Mt could be a good candidate as an antibiotic alternative in weaned pigs.

Con?ict of interest

None declared.

Acknowledgment

This research was supported by the Special Fund for Agro-scienti?c Research in the Public Interest(No.201403047). References

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