Antimicrobial peptide, cLF36, affects performance and intestinal morphology, microflora, junctional proteins, and immune cells in broilers challenged with E. coli

This study investigated the effects of an antimicrobial peptide (AMP), cLF36, on growth performance and the histophysiological changes of the intestine in E. coli-challenged broiler chickens. A total number of 360 day old male chicks were randomly assigned to 4 groups of 6 replicates as follows: T1) negative control diet based on corn-soybean meal without E. coli challenge and additives; T2) positive control diet based on corn-soybean meal and challenged with E. coli without any additives; T3) positive control diet challenged with E. coli and supplemented with 20 mg AMP (cLF36)/kg diet; T4) positive control diet challenged with E. coli and supplemented with 45 mg antibiotic (bacitracin methylene disalicylate)/kg diet. Results showed that T3 improved growth performance and the jejunal morphology of E. coli-challenged chickens similar to those of T4. While antibiotic non-selectively decreased the population of ileal bacteria, AMP increased the population of Lactobacillus spp. and decreased harmful bacteria in the ileum of E. coli-challenged chickens. Supplementing E. coli-challenged chickens with AMP improved the gene expression of immune cells and upregulated the expression of tight junction proteins compared to other challenged groups. In conclusion, although cLF36 beneficially affected growth performance and the intestinal morphology of E. coli-challenged chickens similar to those of the antibiotic group, this AMP drastically improved the intestinal microbiome, immune cells, and junctional proteins compared to other E. coli-challenged birds, and can be nominated as an alternative for growth promoter antibiotics.

Intestinal morphology. Table 2 summarizes the effects of treatments on villi morphology in the jejunum of chickens. Birds challenged with E. coli had lower (P < 0.05) VH, thinner (P < 0.05) VW, and lesser (P < 0.05) VSA compared to the NC birds. At 24 days of age, antibiotic and AMP improved (P < 0.05) VH and VSA compared to control group. Experimental diets had no significant effects on CD and VH/CD at either 10 or 24 days of age.
Bacterial population. The effects of experimental diets on ileal bacterial populations are shown in Table 3. Challenging chickens with E. coli increased (P < 0.05) the population of harmful bacteria (i.e. E. coli and Clostridium spp.) and decreased (P < 0.05) the colonization of beneficial bacteria (i.e. Lactobacillus spp. and Bifidobacterium spp.) compared to the NC group. At d 10, antibiotic decreased (P < 0.05) the population of Lactobacillus spp. and Bifidobacterium spp., while this antibiotic-supplemented diet reduced (P < 0.05) all bacterial populations at d 24 compared to the NC group. Birds supplemented with AMP had the highest Treatment ADG 2 (g) ADFI (g) FCR (g/g) 0-10 11-24 0-24 0-10 11-24 0-24 0-10 11-24 0-24  Table 1. Effects of treatments on growth performance of broiler chickens from 0 to 24 days of age. a,b Values within a column with different letters differ significantly (P < 0.05). 1 NC: negative control group received cornsoybean meal diet without any challenge and additives; PC: positive control group received NC diet inoculated with E. coli without any additives; AMP: PC received group supplemented with 20 mg antimicrobial peptide/ kg diet; Antibiotic: PC received group supplemented with 45 mg antibiotic (bacitracin methylene disalicylate)/ kg diet. 2 ADG: average daily gain; ADFI: average daily feed intake; FCR: feed conversion ratio. 3

Gene expression of immune cells and tight junction proteins.
The effects of experimental diets on gene expression of immune cells and tight junction proteins are shown in Fig. 1. Challenging chickens with E. coli increased (P < 0.05) IL-2 and MUC2 expression, but decreased (P < 0.05) IL-6 expression in the jejunum compared to the NC chickens. Adding AMP to the diet resulted in a reduction (P < 0.05) of IL-2 and MUC2 expression and upregulated (P < 0.05) the expression of IL-6 in the jejunum of E. coli-challenged chickens. Chickens challenged with E. coli had the lowest (P < 0.05) expression pattern of claudin-1 and occludin in the jejunum, while supplementing the diet with antibiotic upregulated (P < 0.05) the expression of tight junction proteins in the jejunum of E. coli-challenged birds. Furthermore, adding antibiotic to the diet of E. coli-challenged chickens did not affect the regulation of immune cells and tight junction proteins in the intestine.

Discussion
Increasing concerns of antibiotic resistance have encouraged scientists to search for antibiotic alternatives having the beneficial effects of antibiotics on growth performance and health criteria while preventing transmission of resistance to microbial populations, like those observed in AMPs. The present study was conducted to assess the potency of a new source of peptides to replace antibiotics in the diet of E. coli-challenged broiler chickens based on data obtained from productive and health attributes. In agreement with previous studies 16,17 , the current findings showed that challenging chickens with E. coli retarded growth and impaired performance, while supplementing the diet with AMP attenuated the negative effects of E. coli and improved FCR similar to those of antibiotic-fed birds. The beneficial effects of AMPs on growth performance of broilers under normal 11,18 and stressful conditions 19 have been observed previously that is consistent with the results of the present study. The morphological characteristics of villi in the jejunum of birds were investigated to find the possible metabolic and physiological action of AMPs on the growth performance of E. coli-challenged birds. The morphology of villi in the small intestine is known as an indicator of gut health 11 . In addition, the intestinal lumen is the main site of nutrients absorption which directly depends on villus morphology and surface area 20 . Morphological analysis in the present study showed that AMP and antibiotic increased VH and VSA in E. coli-challenged birds compared to the control group, which is in agreement with previous studies 11,12 . Consistent with the present results, Liu et al. 21 and Bao et al. 18 reported that supplementing the diet with AMPs extracted from pig intestine and rabbit sacculus rotundus, respectively, improved villus morphology in the duodenum and jejunum of broiler chickens. In general, an increase in VH leads to a greater VSA which increases nutrient absorption from the intestinal lumen 22 and consequently increases growth performance in birds. In the current study, birds supplemented with peptide and antibiotic had better morphological characteristics compared to the control and E. coli-challenged chickens, which resulted in significant improvement in growth performance.
The intestinal microbiome can significantly affect host gut health through various mechanisms such as nutrients absorption, villi morphology, intestinal pH, mucosal immunity, and transporter gene expression 23,24 . In the present study, we examined the effects of treatments on the population of two beneficial (Lactobacillus spp. and Bifidobacterium spp.) and two pathogenic (E. coli and Clostridium spp.) bacteria in the ileum of chickens. Antibiotics decreased the population of all bacteria, while AMP significantly improved the community of beneficial bacteria and reduced the colonization of harmful ones in the ileum, which is consistent with previous studies 25,26 . Bacitracin methylene disalicylate exerts its antibacterial activity on the bacterial ribosome subunit resulting in protein synthesis inhibition 27 . This decreases the number of bacteria and microbial damage in the gut, since this antibiotic has a wide range of antibacterial action and does not distinguish between types of bacteria 27,28 While the definite mechanism by which AMPs can affect the microbial population in the gut has not been found, the suggested mechanism explaining the antimicrobial activity of peptides in controlling the microbial community has been attributed to different surface charges of peptides and pathogens. In detail, AMPs have a net positive charge helping them to electrostatically attach to negatively charged bacterial membranes either to destroy these membranes through physical disruption and/or enzymatic digestion or to pass through the lipid bilayer without exerting any damage. This may interfere with intracellular functions like enzyme activity blockage or inhibiting  Table 3. Effects of treatments on ileal microflora (log 10 CFU g −1 ) in broilers at 10 and 24 days of age. a-c Values within a column with different letters differ significantly (P < 0.05). 1 NC: negative control group received cornsoybean meal diet without any challenge and additives; PC: positive control group received NC diet inoculated with E. coli without any additives; AMP: PC received group supplemented with 20 mg antimicrobial peptide/kg diet; Antibiotic: PC received group supplemented with 45 mg antibiotic (bacitracin methylene disalicylate)/kg diet. 2 SEM: standard error of means (results are given as means (n = 12) for each treatment).
www.nature.com/scientificreports www.nature.com/scientificreports/ protein and nucleic acid synthesis 29 . Our previous results showed that the AMP studied in the current experiment can attach to the bacterial membrane through electrostatic interactions and physically disrupt bacterial bilayer membranes [13][14][15] . Consistent with the previous studies [30][31][32] , the current results showed that AMP can selectively inhibit the growth of bacteria in the gut which may demonstrate the substantial competitive advantage of cLF36 in comparison to antibiotics.
The invasion of pathogenic bacteria into intestinal epithelial cells and mucosal layer induce the gastrointestinal immune cells to produce cytokines which play different roles in the immune responses to pathogens 33 . IL-6 is a multifunctional cytokine that promotes B cell differentiation and T cell activation 34 . Interestingly, IL-6 can play both pro-(i.e. trans-signaling) and anti-(i.e. classic signaling) inflammatory roles under certain Samples were analyzed using qPCR, and GAPDH and β-actin were used as the reference genes. Abbreviations as follows: IL-6, interleukin 6; IL-2, interleukin 2; MUC2, mucin 2; NC, negative control birds received a cornsoybean meal basal diet without AMPs, antibiotic and E. coli challenge; PC, positive control birds received NC diet and orally challenged with one ml of E. coli containing 1 × 10 8 cfu/ml; Antibiotic, birds received PC diet and supplemented with 45 mg antibiotic (bacitracin methylene disalicylate)/kg diet; AMP, birds received PC diet and supplemented with 20 mg peptide/kg diet. The letters on the bar mean show significant difference (P < 0.05). (2019) 9:14176 | https://doi.org/10.1038/s41598-019-50511-7 www.nature.com/scientificreports www.nature.com/scientificreports/ conditions depending on the priority of inflammatory response and the pathophysiological context 35,36 . In agreement with previous findings 29,37 , supplemented AMP upregulated the expression of IL-6 in the jejunum of E. coli-challenged chickens in the current study. It has been shown that AMPs can induce the differentiation of bone marrow-derived dendritic cells in the intestine to secrete IL-6 against pathogenic bacteria to protect the intestinal layer from ulceration 38,39 , which may explain the high expression of IL-6 observed in AMP-supplemented group in the current study.
IL-2 is another key cytokine involved in the cellular immune response by T-cell proliferation and the induction of T regulatory responses, and also in the stimulation of B lymphocytes proliferation and immunoglobulin secretion 40 . In the current study, the expression of IL-2 in the jejunum of chickens was upregulated in response to E. coli challenge, which is in agreement with previous studies in pig and chicken models 41,42 . Supplementation of AMP to the diet downregulated the expression of IL-2 in the jejunum of E. coli-challenged chickens, which may suggest the anti-inflammatory effect of cLF36 in the intestine, which has been reported for other kinds of AMPs 29,37 .
Chickens challenged with E. coli in the present study showed an upregulated expression of MUC2, which is in line with previous reports 43,44 . It was shown that the expression of MUC2 increased in the infectious challenge to secrete more mucin from goblet cells into the intestinal lumen to support the protective layer between the invading bacteria and the epithelial cells 45 . Adding AMP to the diet downregulated the expression of MUC2 in the jejunum of challenged chickens, which may be attributed to the significant inhibitory effect of cLF36 on E. coli colonization in the intestine (as described above), which is in agreement with previous observation 46 . In the current study, antibiotic did not attenuate the negative effects of E. coli on MUC2 expression, which is consistent with previous findings showed that antibiotics may eliminate invading pathogens from the intestinal environment, but be unable to restore the normal circumstances of the intestine after pathogen removal 47 .
It has been well-documented that pathogenic bacteria like E. coli attack the intercellular barriers and disrupt tight junction proteins including claudin-1 and occludin through various mechanisms including chemical degradation by bacterial proteases 48,49 or biochemical alterations of actomyosin ring by phosphorylation 50 or dephosphorylation 51 . This is consistent with the current observations that E. coli-challenged birds showed a drastic decrease in the expression of claudin-1 and occludin in the jejunum. However, AMP upregulated the expression of claudin-1 and occludin in the jejunum of E. coli-challenged chickens, which is in agreement with previous studies 52, 53 reporting AMPs to improve the intestinal epithelial integrity and permeability in the context of E. coli challenge. Although the exact regulatory mechanism of AMPs on tight junction proteins has not been found yet, two possible theories have been suggested. The first theory implies that AMPs may directly activate regulatory proteins (i.e. Rho family) in the intestine of E. coli-challenged mice that increases the expression of junctional proteins and enhances the epithelial barrier function 52,54 . The second theory deals with the antibacterial effects of AMPs on pathogens that decrease the junctional protein disruption and improve the epithelial barrier integrity 55 . Interestingly, antibiotic did not increase the expression of claudin-1 and occludin in the jejunum of E. coli-challenged chickens in the current study, while we expected that antibiotic upregulated the junctional proteins due to the antibacterial nature of antibiotics (based on the second above-mentioned theory regarding AMP's antibacterial effects). In agreement with the present findings, Yi et al. 54 demonstrated that antibiotics did not influence the expression of tight junction proteins after pathogens elimination, maybe due to perturbing the intestinal microbial population. Therefore, the findings of present and previous 52,54 studies may strengthen the possibility of the first theory attributing the beneficial effects of AMPs on epithelial tight junctions to the expression of regulatory proteins, rather than AMPs' antimicrobial effects.
In conclusion, the results of the present study suggest that an antimicrobial peptide, cLF36, derived from camel milk can improve growth performance, ameliorate the intestinal morphology changes, and restore gut microbial balance in chickens challenged with E. coli. In addition, supplemented cLF36 may enhance the immune response to E. coli challenge through regulating the expression of cytokines and mucin. Also, cLF36 can improve the intestinal integrity of E. coli-challenged chickens by upregulating the expression of tight junction proteins. Therefore, cLF36 can be introduced as an alternative to growth enhancer antibiotics, based on its beneficial effects observed in the current study, while more research is required to find other contributing aspects of this AMP.

Methods
All experimental protocols involving animals in the present study were approved by Institutional Animal Care and Use Committee of Ferdowsi University of Mashhad (Protocol number 3/42449) and performed following relevant guidelines and regulations to minimize animal pain, suffering, and distress. Birds, treatments, and experimental design. Three hundred and sixty 1-day-old male chicks (Cobb 500) were purchased from a local commercial hatchery, weighed and randomly placed in floor pens (1.1 m × 1.3 m) covered with wood shavings. Birds were assigned to 4 treatments with 6 replicates containing 15 birds in each replicate. Treatments were as follow: (1) negative control (NC) birds received a corn-soybean meal basal diet without AMPs, antibiotic, and E. coli challenge; (2) positive control (PC) birds received the NC diet and were orally challenged with one ml of E. coli containing 1 × 10 8 cfu/mL; (3) birds received the PC diet supplemented with 20 mg peptide/kg diet (AMP); (4) birds received PC diet and supplemented with 45 mg antibiotic (bacitracin methylene disalicylate)/kg diet (antibiotic). All diets were in mash form and formulated to meet or exceed the minimum requirements of Cobb 500 (Table 4). Birds had free access to feed and water throughout the experiment and the temperature was set at 32 °C for the first 3 days and then gradually reduced to 21  www.nature.com/scientificreports www.nature.com/scientificreports/ AMP production. The AMP used in the present study was derived from camel lactoferrin (cLF) consisting of 42 amino acids which was generated in our lab recently (for more details regarding the peptide cLF chimera production, review previous works [13][14][15] ). Briefly, preparation of recombinant plasmid vector was conducted through transforming synthetic cLFchimera into DH5α bacterium [13][14][15] . Next, the latter bacterial colonies were cultured to harvest plasmid extraction. Then, the recombinant vector was transferred into E. coli (DE3) as an expression host and cultured in 2 mL Luria-Bertani broth (LB) medium for overnight according to standard protocol 56 . In the next step, cultured materials were inoculated in 50 mL LB and incubated at 37 °C with shaking at 200 rpm. Then, isopropyl-β-D-thiogalactopyranoside (IPTG) was added to a final concentration of 1 mM and incubated at 37 °C for 6 h after IPTG induction. Periplasmic protein was collected at different times after IPTG induction (2, 4 and 6 h) according to the method described by de Souza Cândido et al. 57 and analyzed on 12% SDS-PAGE. To purify expressed peptide, Ni-NTA agarose column was used based on the manufacturer's instruction (Thermo, USA). The quality of purified recombinant peptide was assessed on a 12% SDS-PAGE gel electrophoresis, while the Bradford method 58 was used to analyze the quantity of recombinant peptide. More recently, an E. coli expression system 14 was developed in our laboratory that is able to produce 0.42 g/L of recombinant peptide. In the current study, 4 g peptide previously obtained from the recombinant E. coli were purified, lyophilized, and thoroughly mixed with 1 kg soybean meal and then supplemented to the relevant experimental diets. The inhibitory effects of this AMP on various plant 13 and poultry 14 pathogens were recently observed in in vitro.
E. coli challenge. The method of E. coli challenge was explained in details elsewhere 17 with some minor differences. In summary, a suspension of E. coli (ATCC 31616) was cultured on MacConkey agar plates (Merck, Germany) for 24 h at 37 °C, and pink, round medium-sized colonies were picked as E. coli suspect colonies to prepare the inocula. Next, E.coli K99 was inoculated in LB medium and incubated at 37 °C for 24 h. Cell bacteria density was determined in the medium by the subculture of bacteria after making a serial dilution. Bacteria were adjusted to 10 8 cfu/ml by diluting in 0.5% peptone solution. On d 7, chicks were orally challenged with 1 ml of prepared inoculation containing 1 × 10 8 cfu E. coli, while non-challenged chicks received 1 ml of sterile peptone water.
Growth performance. Body weight (BW) and feed remaining of each pen were weighed on days 10 and 24 to measure the average daily gain (ADG), average daily feed intake (ADFI) and feed conversion ratio (FCR) over the specific and entire periods of experiment (0-10, 11-24, and 0-24 days of age). Mortality per pen was recorded daily in order to adjust FCR accordingly.
Sample collection. Two birds from each pen (12 birds/treatment) were randomly selected on days 10 and 24, euthanized by cervical dislocation, the viscera was excised, the intestine was discreetly separated from the whole viscera, and the adherent materials were precisely removed. The ileum was gently pressed to aseptically collect ileal content into sterile tubes for microbiological analysis. A section (about 5 cm) from mid-jejunal tissues was meticulously separated for morphological analysis. A 2 cm section from the mid-jejunum was detached,  RNA extraction and gene expression. The procedure of RNA extraction and gene expression was described previously 61 . In summary, total RNA was extracted from chicken jejunum sampled on day 24 using the total RNA extraction kit (Pars Tous, Iran) following the manufacturer's instructions. Purity and quality of extracted RNA were evaluated using an Epoch microplate spectrophotometer (BioTek, USA) based on 260/230 and 260/280 wavelength ratios, respectively. Genomic DNA was removed using DNase I (Thermo Fisher Scientific, Austin, TX, USA). The complementary DNA (cDNA) was synthetized from 1 µg of total RNA using the Easy cDNA synthesis kit (Pars Tous, Iran) following the manufacturer's protocol. Gene expression of two references (GAPDH and β-actin) and five targets (Interleukin-1 [IL-1], IL-6, mucin2 [MUC2], Claudin-1 [CLDN1], and Occludin [OCLN]) genes were determined by quantitative real-time PCR (qRT-PCR) based on MIQE guidelines 62 . Each reaction was performed in a total volume of 20 μl in duplicate using an ABI 7300 system (Applied Biosystems, Foster City, CA) and 2 × SYBR Green Real Time-PCR master mix (Pars Tous, Iran). Primer details are shown in Table 5. All primers were designed according to MIQE criteria 62 regarding amplification length and intron spanning. All efficiencies were between 90 and 110% and calculated R2 was 0.99 for all reactions. The method 2 −ΔΔCt Ct 63 was used to calculate relative gene expression in relation to the reference genes (GAPDH and β-actin).  Table 5. Sequences of primer pairs used for amplification of the target and reference genes 1 . 1 For each gene the primer sequence for forward and reverse (5′ → 3′), the product size (bp), and the annealing temperature (Ta) in °C are shown. 2 IL-, interleukin-; MUC2, mucin2; CLDN1, claudin1, OCLDN, occludin; GAPDH, Glyceraldehyde 3-phosphate dehydrogenase.