Effects of Lactobacillus acidophilus on the growth performance and intestinal health of broilers challenged with Clostridium perfringens

2018-10-09ZhuiLiWeiweiWangDanLiuandYumingGuo

Journal of Animal Science and Biotechnology 2018年3期

Zhui Li,Weiwei Wang,Dan Liuand Yuming Guo

Abstract Background:Clostridium perfringens is the main etiological agent of necrotic enteritis.Lactobacilli show beneficial effects on intestinal health in infectious disease,but the protective functions of lactobacilli in C.perfringens-infected chickens are scarcely described.This study examined the effects of Lactobacillus acidophilus(L.acidophilus)on the growth performance and intestinal health of broiler chickens challenged with Clostridium perfringens(C.perfringens)over a 28-day period.Using a 2×2 factorial arrangement of treatments,a total of 308 1-day-old male Arbor Acres broiler chicks were included to investigate the effects of Lactobacillus acidophilus(L.acidophilus)on the growth performance and intestinal health of broiler chickens challenged with Clostridium perfringens(C.perfringens)during a 28-day trial.Results:During infection(d 14–21),C.perfringens challenge decreased the average daily gain(P＜ 0.05),and increased feed conversion ratio and the mortality rate(P＜0.05).However,dietary supplementation with L.acidophilus increased the body weight of C.perfringens-infected broilers on d 21(P＜0.05),and tended to decrease the mortality(P=0.061).C.perfringens challenge decreased the villus height(P＜ 0.05),the ratio of villus height to crypt depth(P＜ 0.05)and OCLN(occludin)mRNA expression(P＜0.05),and increased the pro-inflammatory cytokine expression in the spleen and jejunum,the intestinal populations of C.perfringens and Escherichia(P＜0.05),and the serum content of endotoxin(P＜0.05),regardless of L.acidophilus supplementation.In contrast,dietary L.acidophilus reducedthe intestinal lesion score of challenged broilers(P＜0.05),the mRNA expression of pro-inflammatory cytokines,ileal populations of Escherichia and serum endotoxin content(P＜0.05),but increased the intestinal Lactobacillus populations(P＜0.05),irrespective of C.perfringens challenge.Conclusion:Dietary addition of L.acidophilus could improve the intestinal health and reduce the mortality of broilers suffering from necrotic enteritis.

Keywords:Broiler,Clostridium perfringens,Intestine,Lactobacillus acidophilus,Necrotic enteritis

Background

Necrotic enteritis(NE)is a wide-spread poultry disease costing the global poultry industry approximately 2 billion U.S.dollars each year[1].Clostridium perfringens(C.perfringens)type A is the main etiological agent of NE,and causes two forms of disease:a clinical form,with a sudden increase in flock mortality,often without premonitory signs,and a subclinical form,causing suboptimalgrowth performance[2].In-feed antibiotic growth promoters have been used to control NE;however,many countries forbid the dietary use of antimicrobials because of public concern over the emergence of antibiotic-resistant bacteria[3].Outbreaks of NE have been increasing due to the withdrawal of infeed antibiotics[4],indicating a threat to human food security.In recent years,probiotics have been used as an alternative to antibiotics.As probiotics,Lactobicillusspecies have been used to improve the intestinal health and growth performance of poultry.Lactobacilli produce bacteriostatic bacteriocin-like compounds[5]as well as acids,such as lactic acid,which decreases the pH of the gut.Competitive exclusion and antagonism have also been proposed as mechanisms by which lactobacilli species prevent the proliferation of pathogenic bacteria and regulate the intestinal flora[6–8].Previous studies have shown thatLactobacillus acidophilus(L.acidophilus)can inhibit the pathogens[9,10]and modulate the immunity[11].However,it is unclear whether dietaryL.acidophilussupplementation could improve the intestinal health and growth performance of broilers in a NE model.Therefore,we conducted a NE model-based feeding trial to investigate the effects ofL.acidophiluson the intestinal permeability andhistomorphology,cytokine mRNA expression,and microbiota of broiler chickens challenged withC.perfringens.

Methods

Animals and experimental design

A total of 308 1-day-old male Arbor Acres broilers were used to study the effects of additives(without/withL.acidophilus),pathogen challenge(without/withC.perfringenschallenge),and their interactive effects.A 2×2 factorial arrangement was used,with a completely randomized experimental design.All newly hatched healthy birds were weighed and randomly assigned into one of four treatment groups,with seven replicates in each group and 11 birds per replicate.The four groups consisted ofan untreated control(CTL),aL.acidophilusonly supplementation group(LA),aC.perfringensonly challenge group(CLG),and aC.perfringenschallenge group supplemented withL.acidophilus(CLG+LA).All birds were housed in the cages(100 cm×100 cm),and were offered free access to the feed and water throughout the 28-day trial.

Diets and L.acidophilus supplementation

A corn-soybean-meal basal diet in mash form was formulated to meet the nutrient requirements of chickens,as per the recommended feeding standards for broilers in China(NY/T 2004)(Table 1).The ingredients of basal diet were mixed in the same batch to ensure that the experimental diets were identical in composition.A probiotic formulation ofL.acidophilusLAP5(Synlac Material Technology Co.,Nanjing,China)was added to the basal diet at 40 mg/kg,providing 4.0×106cfu/kg of diet.Firstly,we calculated the exact amount of bacteria and basal diet that were needed to produce the experimental diets;secondly,the bacteria were diluted with a small amount of basal diet;thirdly,it would be mixed with the remaining basal diet using the shovel.The experimental diets were produced manually every 4 d.

Table 1 Composition of the basal diet(as-fed basis)

C.perfringens challenge

TheC.perfringenschallenge method used in this study was developed by Dahiya et al.[12],and modified by Liu et al.[13].Briefly,a chickenC.perfringenstype A field strain(CVCC 2030),originally isolated from a clinical case of NE,was obtained from the China Veterinary Culture Collection Center(Beijing,China).C.perfringenswas cultured anaerobically on tryptose-sulfite-cycloserine agar for 18 h at 37°C,and then aseptically inoculated into cooked meat medium and incubated anaerobically for 8 h at 37°C.Birds in the challenge groups were orally gavaged once daily with actively growingC.perfringens(2.0×108cfu/mL,1.0 mL per bird)during d 14–20,while the nonchallenged birds were gavaged with the same volume of sterilized cooked meat medium.

Sample collection

On d 21,one bird in each replicate was randomly selected and humanly euthanized to collect the blood,fresh digesta in the ileum(defined as the region between Meckel’s diverticulum and 2 cm cranial to the ileocaecal junction)and cecum,spleen,and segments in the middle of jejunum(defined as the region from the end of duodenum to the Meckel’s diverticulum).The blood was collected aseptically from the wing vein into normal vacutainers.Serum was then obtained from the blood samples and stored at−20 °C.Spleen and intestinal segments for mRNA isolation were frozen in liquid nitrogen.Fresh ileal digesta(collected from the segments between ileum midpoint and 2 cm proximal to the ileocecal junction)and cecal digesta were collected aseptically and stored at−70 °C to determine the quantitation of theC.perfringens,EscherichiaandLactobacillusspecies populations.

Growth performance

Body weight(BW)and feed intake(FI)for each replicate were measured on d 14,21 and 28.Average daily gain(ADG),average daily feed intake(ADFI),and the feed conversion ratio(FCR)were calculated during the different periods(d 1–14,d 14–21 and d 21–28).

Intestinal lesion score

The small intestine of each bird was excised and subjected to scoring for NE lesions on a scale from 0 to 4,as described by Liu et al.[13].Briefly,0=normal intestinal appearance;0.5=severely congested serosa and mesentery engorged with blood;1=thin-walled and friable intestines with small red petechiae;2=focal necrosis,grey appearance and small amounts of gas production;3=sizable patches of necrosis,gas-filled intestine,and small flecks of blood;and 4=severe extensive necrosis,marked hemorrhage,and large amounts of gas in the intestine.

Intestinal histomorphology

A segment of ileum from each bird was fixed in 4%paraformaldehyde immediately after sacrifice and then embedded in paraffin.Transverse 5-μm sections were stained with hematoxylin and eosin,and then histomorphometrically examined using an Olympus optical microscope and ProgRes CapturePro software(version 2.7;Jenoptik,Jena,Germany).Ten villi were measured in each section and only complete and vertically oriented villi were measured.Villus height was measured from the tip of the villus to the crypt opening and the associate crypt depth was measured from the base of the crypt to the level of the crypt opening.Then the average values of villus height and crypt depth in each section were calculated,respectively.The ratio of villus height to relative crypt depth was calculated from these measurements.

Serum endotoxin content

The barrier integrity and function were evaluated using an indirect method by measuring the serum endotoxin levels. A Quantitative Chromogenic End-point Tachypleus Amebocyte Lysate Endotoxin Detection Kit was used,following the manufacturer’s instructions(Xiamen TAL Experimental Plant Co.,Fujian,China).

Gene expression analysis

Total RNA isolation was carried out by using Trizol reagent(Invitrogen Life Technologies,Carlsbad,CA)according to the manufacturer’s instructions.The concentration and purity of total RNA was monitored using a Nanodrop ND-1000 spectrophotometer(Thermo Fisher Scientific Inc.,Waltham,MA,USA).One microgram of total RNA was reverse transcribed by a reverse transcription kit(Takara Bio Inc.)according to the manufacturer’s instructions.Reverse transcription was performed at 42 °C for 2 min,37 °C for 15 min,followed by heat inactivation for 5 s at 85°C.All of the cDNA preparations were stored frozen at 20°C until further use.On d 21,the mRNA expression ofIL-1β,IL-8,interferon gamma(IFN-γ),tumor necrosis factor alpha(TNF-α),andIL-10in the spleen and jejunum andmucin 2(MUC2),claudin 1(CLDN1),occludin(OCLN)andzonula occludens 1(ZO-1)in the jejunum were measured by quantitative real-time PCR(qRT-PCR)analysis.Gene specific primer sequences are shown in Table 2.The qRT-PCR assay was performed using a 7500-fluorescence detection system (Applied Biosystems,Foster City,California)and a commercial SYBR-Green PCR kit(Takara Bio Inc.,Ostu,Japan).The following thermal cycler conditions were used:95°C for 30 s,40 cycles of 95 °C for 5 s and 60 °C for 34 s.At the end of each run,melting curve analysis and subsequent agarose gel electrophoresis of the PCR products were carried out to confirm the amplification specificity.The β-actinwas used as the housekeeping gene,and the data of relative gene expression were analyzed using the 2−ΔΔCtmethod as previously described(Livak and Schmittgen,[14]).The amplifying efficiency of the qRT-PCR primers for each target gene is between 90%and 110%.

Ileal and cecal microbiota enumeration

The populations ofC.perfringens,Escherichiasubgroup,andLactobacillussubgroup species in the digesta were detected by absolute qRT-PCR,as described previously[15,16],with some modification.Briefly,genomic DNA was isolated from about 200 mg of digesta from the ileum and caecum using a QIAamp DNA Stool Mini Kit(Qiagen Inc.,Valencia,CA).Extracted DNA was stored at−70 °C until analysis.Standard curves for qRT-PCR were prepared by normal PCR amplification using DNA extracted from pure bacterial cultures to produce a high concentration of the target DNA.CompetentEscherichia coliDH5α(Takara Bio Inc.,Japan)were used to generate plasmid standards.The PCR products were purified using a PCR purification kit (Biomed GeneTechnologies,Beijing,China),and then cloned into pCR2.1 using a TA cloning kit(Invitrogen Corporation,Carlsbad,CA),as per the manufacturer’s protocol.Purified insert-containing plasmids were quantified using a Nanodrop ND-1000 spectrophotometer(Thermo Fisher Scientific Inc.,Waltham,MA),and the number of target gene copies was calculated using the following formula,as described previously[17]:

Table 2 Quantitative real-time PCR primer sequences

Standard curve was constructed by the ten-fold serial dilutions of plasmid DNA.Genomic DNA from ileal and cecal samples was used as a template for absolute qRTPCR using a 7500 fluorescence detection system(Applied Biosystems,Foster City,CA)according to optimized PCR protocols(SYBR-Premix Ex Taq,Takara Bio Inc.,Japan).The qRT-PCR primers were the same as used for normal PCR amplification(Table 3).The data were presented as log10gene copies/g of intestinal digesta.

Statistical analysis

All data were analyzed with SPSS version 20.0(SPSS Inc.,Chicago,IL).A significance level of 0.05 was used.The data of mortality and intestinal lesion score were analyzed by one-way ANOVA,and were subjected togrouped table of the GraphPad Prism 5(GraphPad Software,Inc.,CA,USA).Results in figures were represented as mean±SE of 7 replicates,with 11 birds per replicate.Other data were analyzed by two-factorial analysis of variance to examine the main effects of probiotic and challenge,and their interaction using general linear model procedure SPSS 20.0.When a significant interaction between the main effects was observed,Duncan’s multiple comparison was used to compare the differences among the four groups.Results in the tables were given as the mean and pooled SEM.

Table 3 The sequence of 16S rRNA quantitative real-time PCR primers used to quantify intestinal bacteria

Results

Growth performance and mortality

The growth performance of broiler chickens was not significantly affected by dietaryL.acidophilusaddition during the d 0–14(P＞0.05)(data not shown).However,as shown in Table 4,C.perfringenschallenge decreased the ADG(P＜0.05)and increased the FCR(P＜0.05)of broilers during d 14–21.An interaction between the effectsC.perfringensandL.acidophilustreatment on BW of broilers was observed on d 21(P＜0.05),andL.acidophilustreatment significantly increased the BW of broilers when challenged(P＜0.05).No deaths were observed in non-challenged broilers(CTL and LA groups)during the experimental period,whileC.perfringenschallenge significantly increased the mortality rate(P＜0.05)of broilers from d 14 to 21,andL.acidophilusaddition tended to decrease the mortality rate(P=0.061)(Fig.1).The growth performance of broilers during d 21–28 was not significantly affected byC.perfringenschallenge orL.acidophilussupplementation(P＞0.05,data not shown).