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http:// www.jstage.jst.go.jp / browse / jpsa doi:10.2141/ jpsa.0130137 Copyright Ⓒ 2014, Japan Poultry Science Association. Influence of Propolis Residue on the Bacterial Flora in the Cecum of Nanbu Kashiwa Kazumi Kita1, Ito R. Ken1, Chinami Akamine1, Wataru Kawada2, Yoichiro Shimura2 and Tamio Inamoto2 1 2 Faculty of Agriculture, Iwate University, Morioka, Iwate 020-8550, Japan Faculty of Bioresource Sciences, Akita Prefectural University, Shimoshinjo, Akita 010-0195, Japan Since the growth promoter effect of antibiotics was found, many kinds of antibiotics have been used as feed additives for the improvement of growth performance. Since January 1, 2006, however, European Parliament prohibited the use of antibiotics as feed additives for promoting animal growth because of biosecurity threats arising from the increasing resistance of pathogens to antibiotics. Thereafter, various natural substrates having the growth promoter effect have been attempted to use as feed additives instead of antibiotics. Honeybees collect propolis from the cracks in the bark of trees, and it has the versatile pharmacological activities including antibacterial effect. Therefore, the present study aimed to examine the influence of propolis residue, which was the residue after ethanol extraction, on the cecal bacterial flora in the meat-type chicken, Nanbu Kashiwa. Twenty-eight-d-old female Nanbu Kashiwa were given an ordinary diet for 90 days. During last 10 days, an experimental diet including 2% of propolis residue was provided. At the end of experimental period, ceca were removed and DNA was isolated from cecal content. The V3 region of the bacterial 16S rRNA gene was amplified by PCR using the 357F-GC and 518R primers. To indentify the bacterial diversity, denaturing gradient gel electrophoresis (DGGE) was applied, and the optical intensity of DGGE bands was determined. The major bands were excised, and DNA was reamplified by PCR. The sequencing data were analyzed by the Basic Local Alignment Search Tool (BLAST) of DNA Data Bank of Japan. The bacteria identified by PCR-DGGE and BLAST were Lactobacillus aviaries, Lactobacillus, Olsenella, Coriobacterium, Paraprevotella, Prevotellaceae, Clostridiaceae and Bacteroidaceae. The DGGE band of which the optical intensity of the propolis residue group was lower than the control group was the family Prevotellaceae. The successful use of propolis residue to reduce the family Prevotellaceae showed the possibility to use propolis residue as alternative feed additives. Key words: 16S rRNA, bacterial flora, cecum, denaturing gradient gel electrophoresis, Nanbu Kashiwa, propolis residue J. Poult. Sci., 51: 275-280, 2014 Introduction In 1940’s, the growth promoter effect of antibiotics was observed in animals fed dried mycelia of Streptomyces aureofaciens containing chlortetracycline, and the mechanism of action of antibiotics as growth promoters is related to interactions with intestinal microbial population (Dibner and Richards, 2005; Niewold, 2007). Thereafter, many kinds of antibiotics have been used as feed additives for the improvement of growth performance. However, contemporary biosecurity threats arising from the increasing resistance of pathogens to antibiotics elicited a call for the ban of Received: July 16, 2013, Accepted: October 18, 2013 Released Online Advance Publication: November 25, 2013 Correspondence: Dr. K. Kita, Faculty of Agriculture, Iwate University, Morioka, Iwate 020-8550, Japan. (E-mail: [email protected]) antibiotic growth promoters (Barton, 2000; Snel et al., 2002). In 2003, Regulation 1831/2003 on additives for use in animal nutrition was stated by the European Parliament. This regulation stated that antibiotics used as feed additives for promoting animal growth will have been prohibited until December 31, 2005 (Castanon, 2007). Thereafter, various natural substrates having the growth promoter effect have been attempted to use as feed additives instead of antibiotics. Honeybees (Apis mellifera L.) collect the resin from the cracks in the bark of trees and leaf buds. The resin is called propolis, sometimes also referred to bee glue. Propolis is defined by U.S. National Library of Medicine (2013) as a resinous substance obtained from beehives that is used traditionally as an antimicrobial and that is a heterogeneous mixture of many substances. Because of its popularity in folk medicine, propolis has become the subject of intense Journal of Poultry Science, 51 (3) 276 pharmacological and chemical studies so far. A large number of studies have proven its versatile pharmacological activities: antibacterial, antifungal, antiviral, antioxidant, anticancer and anti-inflammatory activities (Grange and Davey, 1990; Kujumgiev et al., 1999; Lotfy, 2006; Sforcin, 2007; Viuda-Martos et al., 2008; Mavri et al., 2012; Sawicka et al., 2012). However, although it was reported that the inclusion of propolis in poultry feeds affected some hematological parameters, i.e. erythrocyte count, IgG concentration and IgM concentration (Cetin et al., 2010), the influence of dietary propolis on bacterial flora in the cecum has not been examined so far. Moreover, in most studies, ethanol extract was used as propolis, and the antibacterial effect of propolis residue after ethanol extraction has not been investigated. The PCR-DGGE analysis was employed to classify the bacteria in the cecum of chickens. The DGGE is an electrophoretic method to identify single base change in a segment of DNA. In a denaturing gradient acrylamide gel, double-strand DNA is subjected to an increasing denaturant environment and will melt in discrete segment called melting domeins. The melting temperature of these domeins is sequence specific. When the melting temperature of the lowest melting domein is reached, the DNA will become partially melted, creating branched molecules. Partial melting of the DNA reduces its mobility in a polyacrylamide gel. Since the melting temperature of a particular melting domein is sequence-specific, DNA containing different sequences will encounter mobility shifts at different position in the gel. In the present study, therefore, the influence of propolis residue after ethanol extraction in poultry feeds on cecal bacterial flora in meat-type chickens was examined by PCRDGGE. Materials and Methods Animals and Experimental Diets Twenty-eight-d-old female meat-type chickens ‘Nanbu Kashiwa’ ((Rhode Island Red×White Plymouth Rock) × (Game Fowl× (Colored Cornish×Iwate Native Fowl))) was purchased from a prefectural research center (Animal Industry Research Institute, Iwate Agricultural Research Center, Takizawa, Iwate, Japan), and they were given a diet for meat-type chickens (grain 65%, oil seed meals 21%, fish meal 6%, others 8%; CP 18%, ME 3,300 kcal/kg) for 90 days. Chickens were allowed free access to water. During last 10 days of experimental period, an experimental diet including propolis residue was fed to 9 chickens. Propolis residue (Kakudate Apiary, Inc., Hachimantai, Iwate, Japan) was added to the diet at the level of 2%. Propolis residue used in the present study was immersed in edible alcohol (ethanol) for 2 years and the residue after alcohol extraction was used. As a control, the same feed for meat-type chickens was fed to 9 birds. All chickens were raised in a rural farm (Ludens Farm Inc., Nishine, Iwate, Japan). At the end of experimental period, ceca were removed and stored at −30℃ until analysis. Animal care was in compliance with applicable guidelines from the Iwate University Animal Care and Use Committee. Linearity of PCR-amplified DNA to Bacteria Quantity In the present study, PCR-amplified DNA of cecal bacteria was supposed to be quantified by using a commercial image analyzer (Gel-Pro Analyzer, version 3.1, Nippon Roper Co. Ltd., Tokyo, Japan). To confirm the linearity of PCRamplified DNA to bacteria quantity, DNA purified from Campylobacter jejuni was amplified by PCR and quantified by GelPro Analyzer. Campylobacter jejuni were incubated at 40℃ for 48 h in micro anaerobic nutrient agar called by brain heart infusion agar (PEALCORE®, Eiken Chemical Co., Ltd., Tochigi, Japan) including defibrinated horse blood (Nippon Bio-Test Laboratories Co., Ltd., Tokyo, Japan) and Campylobacter selective supplement (Butzler) (SR0085E, Oxoid Co., Ltd., Basingstoke, Hants, England). Micro anaerobic nutrient agar plates were maintained in a BBLTM GasPak 100TM Anaerobic System (Becton Dickinson Co., Ltd., Franklin Lakes, NJ. USA) with AnaeroPack®-MicroAero (Mistubishi gas chemical Co., Ltd., Tokyo, Japan). To collect campylobacter, 4 mL of Dulbecco’s phosphate-buffered saline (DPBS, Gibco®, Life Technologies Japan Ltd., Tokyo, Japan) was put onto agar plates, and 3 mL of suspension containing campylobacter was recovered. The absorbance of campylobacter suspension was measured at 600 nm, and the suspension was diluted with DPBS at the level of 0.05, 0.1, 0.2, 0.4, and 0.8 of absorbance (600 nm). DNA was extracted from suspension by using ISOPLANT II DNA extraction kit (Nippon Gene Co. Ltd., Tokyo, Japan) in accordance with the manufacturer’s protocol. Total DNA was dissolved in 200 μL of TE (10 mM Tris-HCl (pH 7.5), 1 mM EDTA) buffer. Using the extracted bacterial DNA, 16S rRNA gene amplification was performed by PCR using the following primers to amplify the V3 region of the bacterial 16S rRNA gene: 357F-GC, 5′ -CGC CCG CCG CGC GCG GCG GGC GGG GCG GGG GCA CGG GGG GCC TAC GGG AGG CAG CAG-3′ ; 357F primer was used to add a 40-nucleotide GC rich sequence at the 5′end) and 518R (5′ GTA TTA CCG CGG CTG CTG G-3′ ; Muyzer et al., 1993). The concentration of each primer was 10 pmol/μl. The 20 μl using GoTaq Green Master Mix (Promega, Madison, WI, USA) was mixed with 2 μl of primer solution and 5 μl of bacterial DNA solution (50 μg/ml). The PCR reactions were performed under the following conditions: 94℃ for 4 min; 20 cycles of 94℃ for 0.5 min, 65-56℃ for 0.5 min, and 72℃ for 0.5 min, with a stepwise decrease of 1℃ after every second cycle; 10 cycles at 94℃ for 0.5 min, 55℃ for 0.5 min, and 72℃ for 1 min; 1 cycle at 72℃ for 4 min, and 4℃ for storage. The presence and yield of PCR products were assessed using agarose gel electrophoresis. After PCR, optical density of amplified DNA bands was measured by Gel-Pro Analyzer. The regression equation of optical intensity of PCR-amplified DNA bands to the absorbance at 600 nm was calculated using the General Linear Model Procedures of SAS (SAS/STAT version 6) (SAS Institute, 1999). Kita et al.: Propolis Residue and Cecal Bacterial Flora PCR-DGGE of Cecal Bacteria Flora of Chickens Given Dietary Propolis Residue Before isolation of total DNA from cecal content, less than 1 g of cecal content was washed by passing through a strong nylon mesh (Cell strainers, 100 μm mesh, BD Falcon®, Japan BD Co. Ltd., Fukushima, Japan) with DPBS. After passing through the mesh, samples were centrifuged at 3, 300×g, 4℃ for 10 min. The supernatant was discarded, and the pellet was suspended in 35 ml of DPBS. The suspension was centrifuged, and the supernatant was discarded. The pellet was suspended in 35 ml of TE buffer and centrifuged. After discarding the supernatant, the pellet was suspended in 10 ml of TE buffer. The 1 ml aliquots of suspension were centrifuged at 9,300×g , 4℃ for 10 min, and the supernatant was discarded. Washed cecal content was stored at −80℃. Total DNA was extracted from washed cecal content by using ISOPLANT II DNA extraction kit in accordance with the manufacturer’s protocol. Total DNA was dissolved in 200 μL of TE buffer. Using the extracted bacterial DNA, 16S rRNA gene amplification was performed by PCR as described above. The primers to amplify the V3 region of the bacterial 16S rRNA gene were 357F-GC and 518R. The presence and yield of PCR products were assessed using agarose gel electrophoresis. In the present study, to indentify the bacterial diversity, denaturing gradient gel electrophoresis (DGGE) was applied. The DGGE analysis was conducted in accordance with the manufacturer’s protocol of The DCodeTM Universal Mutation Detection System (Bio-Rad, Hercules, CA, USA). Polyacrylamide gel (8%) containing gradients of 20 to 60% denaturant (100% denaturant corresponds to 7 M urea and 40% deionized formamide) was prepared using a gradient maker (Model 475 Gradient Delivery System, Bio-Rad). The gel to which DNA samples (5 μL of PCR reactant and 2 μL of 70% glycerol in each lane) was applied was run for 5 h at 130 V at 60℃ in 1× TAE (40 mM Tris-acetate, 1 mM EDTA; pH 8.0) buffer. DGGE marker II (Nippon Gene) was used as a marker. After electrophoresis, the gel was stained with SYBR Gold (Invitrogen, Carlsbad, CA, USA) nucleic acid gel stain, and band images were detected using a luminoimage analyzer (LAS-4000, FUJIFILM Co. Ltd., Tokyo, Japan). The optical intensity of DGGE bands was analyzed by using a commercial image analyzer. The major bands were excised with a sterile razor, and the DNA was eluted by incubating a gel slice in TE buffer at 4℃ overnight. The eluted DNA was purified using Wizard SV Gel and a PCR Clean-UP System (Promega), and then used as a template for the following PCR reamplification with primers 357F (5′ -CCT ACG GGA GGC AGC AG-3′ ) and 518R under the conditions described above. The reamplified 16S rRNA gene was ligated into the pGEM-T Easy Vector (Promega), and then the plasmids were introduced into Escherichia coli DH5α. The 16S rRNA gene on the purified plasmids was sequenced using primers M13F and M13R (Yanisch-Perron et al., 1985) with the BigDye Terminator Cycle Sequencing Kit (Applied Biosystems, Foster City, CA, 277 USA) according to the manufacturer’s instructions. Sequences were then analyzed using an Applied Biosystems 3100 Genetic Analyzer. The sequencing data were analyzed by BLAST of DDBJ (http://blast.ddbj.nig.ac.jp/blastn?lang= ja) on 16S rRNA gene for Bacteria and Archeae database. Statistical Analyses Results are expressed as means±SE. Statistical analysis of data was performed by one-way ANOVA followed by student’s t-test using the General Linear Model Procedures of SAS (SAS/STAT version 6) (SAS Institute, 1989). Results Linearity of PCR-amplified DNA to Bacteria Quantity The regression equation of optical intensity of PCRamplified DNA bands (Y) to the absorbance at 600 nm (X) was Y=78.1X+96.1 (Fig. 1). The absorbance at 600 nm of the suspension of Campylobacter jejuni was significantly and linearly related to the optical intensity of PCR-amplified DNA bands. PCR-DGGE of Cecal Bacteria Flora of Chickens Given Dietary Propolis Residue PCR-DGGE profiles of 16S rRNA gene fragments derived from bacterial flora in the cecum of meat-type chickens (Nanbu Kashiwa) fed a diet containing propolis residue were represented in Fig. 2. The DGGE bands with arrows and numbers had the significant difference in the optical intensity of bands between propolis residue and control groups. The values for optical intensity of DGGE bands of 16S rRNA bacterial gene with significant difference between propolis residue and control groups are shown in Table 1. The optical intensity of 2 bands (band No. 1 and 3) of chickens fed a diet containing propolis residue was higher than that of birds fed a control diet. On the other hand, the optical intensity of a band (band No. 2) of chickens given the control diet was higher than that of birds given propolis residue. The sequencing data of 16S rRNA gene were analyzed by a BLAST search of NCBI database (16S ribosomal RNA Fig. 1. The regression equation of optical intensity of PCR-amplified DNA bands (Y) to the absorbance at 600 nm (X). Circles are means±SE. n=3 (one missing value at the lever of 0.8 of absorbance). Journal of Poultry Science, 51 (3) 278 PCR-DGGE profiles of 16S rRNA gene fragments derived from bacterial flora in the cecum of chickens (Nanbu Kashiwa) fed a diet containing propolis residue. DNA samples were extracted from 9 samples of cecum content. The 16S rRNA gene fragment was amplified by universal PCR primer sets (forward, 357F-GC; reverse, 518R). Six samples were randomly selected from 9 samples of PCR products. The DGGE bands with arrows and numbers had the significant difference in the optical intensity analyzed by an image analyzer (GelPro analyzer) between propolis residue and control groups. Fig. 2. The optical intensity of DGGE bands of 16S rRNA gene of bacteria in the cecum of meat-type chickens (Nanbu Kashiwa) fed a diet containing propolis residue Table 1. DGGE bands 1 2 3 Dietary propolis residue level 0% 2% 32 . 0b,*±7 . 0 42 . 7a±6 . 9 4 . 4b±1 . 3 181 . 8a,*±25 22 . 4b,*±4 . 7 18 . 5a,*±3 . 7 The values were the optical intensity measured by a commercial image analyzer (GelPro Analyzer) with the significant difference between propolis residue and control groups. Means not sharing a common superscript letters within the same row are significantly different at P<0.05. Values are means±SE from 6 chickens in each group. * One missing value. gene (Bacteria and Archea)), and when the sequence was identical to the database at the level of more than 95% and 90-95%, the rank was regarded as family and genus, respectively. DNA sequencing results of DGGE bands were deposited in DDBJ database under accession numbers AB817937 (uncultured Lactobacillus sp.), AB817938 (uncultured Bacteroidaceae bacterium), AB817939 (uncultured Lactobacillus sp.), AB817940 (uncultured Clostridiaceae bacterium), AB817941 (uncultured Lactobacillus sp.), AB817942 (uncultured Prevotellaceae bacterium), AB817943 (uncul- Sequence analysis of 16S rRNA gene of bacteria in the cecum of meat-type chickens (Nanbu Kashiwa) given dietary propolis residue Table 2. DGGE bands Closet relative Rank Identity (%) 1 2 3 Clostridiaceae Prevotellaceae Coriobacterium family family genus 92 93 97 The sequencing data were analyzed by a Blast search of Data Bank of Japan (DDBJ). When the bacterial sequence was identical to the database at the level of more than 95% and 90-95%, the rank was regarded as family and genus, respectively. tured Clostridium sp.), AB817944 (uncultured Olsenella sp.), AB817945 (uncultured Olsenella sp.), AB817946 (uncultured Coriobacterium sp.), AB817947 (uncultured Bacteroidaceae bacterium), AB817948 (uncultured Paraprevotella sp.), AB817949 (uncultured Bacteroidaceae bacterium), AB817950 (uncultured Prevotellaceae bacterium), AB817951 (uncultured Bacteroidaceae bacterium). The bacteria identified in the present study were Lactobacillus aviaries (identity 100%), Lactobacillus (identity 100%), Olsenella (identity 99%), Coriobacterium (identity 97%), Paraprevotella (identity 96%), Prevotellaceae (identity 93%), Clostridiaceae (identity 92%) and Bacteroidaceae (identity 92%). Sequence analysis of 16S rRNA gene with significant difference in the optical intensity of DGGE bands between propolis residue and control groups is shown in Kita et al.: Propolis Residue and Cecal Bacterial Flora Table 2. Two bands (band No. 1 and 3) of which the optical intensity of the propolis residue group was higher than that of chickens fed a control diet were identified to the genus Coriobacteriumand the family Clostridiaceae, respectively. On the other hand, a band (band No. 2) of which the optical intensity of the propolis residue groups was lower than that of the control group was the family Prevotellaceae. Discussion As represented in Fig. 2, the regression equation of optical intensity of PCR bands (Y) to the absorbance at 600 nm (X) was calculated as Y=78.1X+96.1. This equation confirmed that PCR-DGGE followed by the measurement of optical intensity of amplified DNA bands using GelPro Analyzer can be a tool to quantify the amount of cecal bacteria of chickens. The PCR-DGGE analysis followed by DNA sequence analysis and BLAST search revealed the presence of various types of bacteria, i. e. Lactobacillus, Olsenella, Coriobacterium, Paraprevotella, Prevotellaceae, Clostridiaceae and Bacteroidaceae. The presence of Lactobacillus, Clostridiaceae and Bacteroidaceae in the chicken was also reported by other researchers (Geier et al., 2009a, b; Lumpkins et al., 2010), which was in good agreement to our observation. Since the PCR-DGGE analysis was developed by Muyzer et al. (1993) using a 16S rRNA gene of bacteria, this method has been beneficially used in the field of poultry science. At the beginning, the DGGE analysis was applied to examine the characteristics of bacteria. Johnston and Fernando (1995) analyzed the genetic variability between species and strains of Eimeria spp. This method was also applied to evaluate the subtype of Campylobacter jejuni isolated from poultry (Nielsen et al., 2000) and to examine the influence of growth on the spatial variation of the intestinal bacterial community of broiler chickens (Van der Wielen et al., 2002). Thereafter, the influence of dietary manipulation (Thompson et al., 2008; Geier et al., 2009a, b; Janczyk et al., 2009; Nava et al., 2009) and feed additives like antibiotics (Knarreborg et al., 2002; Pedroso et al., 2006; Zhou et al., 2007) on the microbial flora in the intestine of chickens was studied. In the present study, propolis residue was used as feed additives to anticipate the antibacterial effect against intestinal microbial flora. As expected, dietary propolis residue successfully reduced the family Prenvotellaceae (Tables 1 and 2). The successful use of propolis residue to reduce the family Prevotellaceae showed the possibility to use propolis residue as alternative feed additives. As represented in Tables 1 and 2, in contradiction to Prevotellaceae, gram-possitive bacteria Clostridiaceae and Coliobacterium were significantly increased by dietary propolis residue additive treatment. As Kačániová et al. (2012) reported that feeding of propolis to chickens increased enterococcus and lactobacillus in excreta, propolis residue seems to have the potency to increase the growth of some kinds of bacteria. Boyanova et al. (2006) reported that Bulgarian propolis was highly active against the anaerobic bacteria including 279 Prevotellaceae. As shown in Tables 1 and 2, the antibacterial effect against the family Prevotellaceae was also found in the propolis residue collected from Iwate prefecture in Japan. Interestingly, in spite of the great differences in the chemical composition of propolis from different geographic locations, all samples exhibited similar antibacterial activity (Kujumgiev et al., 1999), and this finding points out that analyses of chemical composition of propolis to identify one individual substance or a particular substance class could not be responsible for the antibacterial activity (Cetin et al., 2010). However, the propolis residue used in the present study was collected in the region of ‘Hachimantai’ of Iwate prefecture of Japan. In this area, the prior vegetation is Japanese Beech, in which several antibiotics have been found (Taguri et al., 2004, 2006). Therefore, some kinds of antibacterial in Japanese Beech seem to be affective to the bacteria growth, and this issue needed to be elucidated in the future. 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