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Effects of a Campylobacter jejuni Infection on the Development of the Intestinal Microflora of Broiler Chickens
ENVIRONMENT, WELL-BEING, AND BEHAVIOR Effects of a Campylobacter jejuni Infection on the Development of the Intestinal Microflora of Broiler Chickens C. H. Johansen,*† L. Bjerrum,*1 K. Finster,† and K. Pedersen* *Danish Institute for Food and Veterinary Research, Hangoevej 2, DK-8200 Aarhus N, Denmark; and †University of Aarhus, Institute of Biological Sciences, Department of Microbiology, Bldg. 540, DK-8000 Aarhus C, Denmark ant gradient gel electrophoresis (DGGE) profiles generated from cecal and ileal contents revealed several DGGE bands that were present in the control chickens, but not in the chickens colonized with C. jejuni. Some of these DGGE bands could be affiliated with Lactobacillus reuteri, Clostridium perfringens, and the genus Klebsiella.
ABSTRACT The effect of a Campylobacter jejuni colonization on the development of the microflora of the cecum and the ileum of broiler chickens was studied using molecular methods. The infection did affect the development and complexity of the microbial communities of the ceca, but we found no permanent effect of a C. jejuni infection on the ileal microflora of the broilers. In addition, denatur-
Key words: broiler chicken, Campylobacter, gut microflora, denaturant gradient gel electrophoresis 2006 Poultry Science 85:579–587
fact that far from all bacteria are culturable in vitro, which has been discovered after the introduction of culture-independent molecular methods in microbial ecology (Torsvik et al., 1996; Tannock et al., 2000). Over the last decade, denaturant gradient gel electrophoresis (DGGE; Muyzer et al., 1993) has been used successfully to investigate the diversity of complex bacterial communities. This method has been widely used to monitor temporal and spacial changes in community structure in response to changes in environmental parameters (Simpson et al., 2000; Cocolin et al., 2001; Casamayor et al., 2002). The aim of the present study was to examine and identify differences in the development of the bacterial community of the ceca and ilea of 2-, 9- and 17-d-old broiler chickens experimentally infected at 1 d of age with C. jejuni compared with uninfected control chickens. This was done by comparing DGGE banding patterns from the different groups of chickens.
INTRODUCTION Campylobacter jejuni, a natural inhabitant of the gastrointestinal tract of chickens and other birds, is currently the most common cause of acute bacterial gastroenteritis (Tauxe, 1992, 2002; CDC, 2004). Increasing evidence suggests a strong association between poultry and human illness caused by C. jejuni (Pearson et al., 2000; Vellinga and Van Loock, 2002; Stern et al., 2003). Levels of Campylobacter in the cecum of infected chickens tend to be high, and typically reported cfu values are 108 to 109/g of content (Heres et al., 2003). Preventing contamination of poultry and poultry products with foodborne human pathogens such as Campylobacter is a challenge for commercial producers worldwide. It is well recognized that the gastrointestinal microbial community of broiler chickens plays an important role in the growth and health of the birds and that the intestinal bacterial composition can be affected by various factors such as diet, stress, probiotics, and antibiotics (Coates et al., 1963; Netherwood et al., 1999; Craven, 2000; Apajalahti et al., 2001; Knarreborg et al., 2002). Generally, little is known about the composition and dynamics of the intestinal microbial community of chickens. Studies on the composition of the intestinal microflora have traditionally been carried out using classical microbiological culture methods (Salanitro et al., 1974; Barnes, 1979; Coloe et al., 1984). However, culture-based diversity studies are biased by the
MATERIALS AND METHODS Animals, Experimental Infection, and Sampling The study was carried out with 60 conventional broiler chickens (Ross 208) purchased at 1 d of age from a local hatchery (Fællesrugeriet, Randers, Denmark). The chickens were transferred directly from the hatchery to the experimental unit, where they were housed in isolators (HM 1500, Montair Andersen B.V., Sevenum, The Netherlands). Prior to the experimental infection, transport boxes and feed and water samples from each isolator were analyzed for the presence of Campylobacter by cultivation. The day-
2006 Poultry Science Association, Inc. Received September 28, 2005. Accepted December 19, 2005. 1 Corresponding author: [email protected]
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old chickens were checked for Campylobacter by cloacal swabs. One group of 30, 1-d-old chickens was experimentally infected orally with 6 × 106 cfu of C. jejuni strain DVI-sc181. The control group of 30 chickens was given a 0.9% NaCl solution. The chickens had access to feed and water ad libitum. They were fed conventional broiler feed without antimicrobial additives (Danish Institute of Agricultural Sciences, Foulum, Denmark). From each isolator, 10 birds were randomly selected at ages 2, 9, and 17 d and killed by decapitation. The number of C. jejuni (cfu) in the cecal and the ileal contents of the chickens was quantified by the spread-plate method on modified charcoal cefoperazone deoxycholate agar (Oxoid, Basingstoke, UK). In addition, samples from the ceca and ilea were collected in Eppendorf tubes for a 16S rDNA-based study of the microbial community and stored at −80°C until further processing.
DNA Extraction Deoxyribonucleic acid was extracted from the cecal and the ileal contents of broilers. For DNA extraction, 250 mg of intestinal content was suspended in 600 L of PBS. The suspension was mixed thoroughly and centrifuged at 200 × g for 2 min. The supernatant was transferred to a new Eppendorf tube and centrifuged at 12,000 × g for 5 min. Subsequently, the pellet was resuspended in 570 L of TEbuffer (10 mM Tris, 1 mM EDTA; pH 8.0) and transferred to a screw-cap tube containing 400 g of sterile zirconium beads (diameter = 0.1 mm; Biospec Products, Bartlesville, OK). The cells were lysed by physical disruption for 3.5 min in a minibead beater (Techtum, Umea˚, Sweden) on high speed. After homogenization, the sample was centrifuged, and the supernatant was transferred to a new tube to which 100 L of 5 M NaCl and 80 L of CTAB/NaCl [10% CTAB (wt/vol) = hexadecyltrimethyl ammonium bromide; Sigma-Aldrich, St. Louis, MO), 0.7 M NaCl) was added. The suspension was mixed thoroughly and placed in a water bath for 10 min at 65°C. The sample was extracted 3 times with 0.7 mL of chloroform:isoamyl alcohol (24:1), 0.7 mL of phenol:chloroform:isoamyl alcohol (25:24:1), and 0.6 mL of chloroform:isoamyl alcohol (24:1), respectively (Sigma-Aldrich). The DNA was precipitated with 99.9% cold ethanol. Finally, the DNA was dissolved in 80 L of TE-buffer and stored at −20°C until further use.
PCR Amplification with Universal 16S rDNA Primers Polymerase chain reaction amplifications of total bacterial community DNA were performed using primer pair HDA1-GC (5′-CGC CCG GGG CGC GCC CCG GGC GGG GCG GGG GCA CGG GGG GAC TCC TAC GGG AGG CAG CAG T-3′; the GC-clamp is in boldface) and HDA2 (5′-GTA TTA CCG CGG CTG CTG GCA C-3′; Walter et al., 2000). These primers amplify the V2-V3 region of the 16S rDNA gene (position 339-539 in Escherichia coli), resulting in fragments of 200 bp. The following thermocycling program was used: 94°C for 4.5 min; 30 cycles of
94°C for 30 s, 56°C for 30 s, and 68°C for 1 min; and finally, 68°C for 7 min (Walter et al., 2000). Polymerase chain reaction was performed in 0.2-mL tubes using a Peltier Thermal Cycler 200 (MJ Research Inc., Watertown, MA). The reaction mixture (50 L) contained 1 × SuperTaq reaction buffer, 200 M of each dNTP (Amersham Biosciences, Piscataway, NJ), 20 pmol of each primer (DNA-Technology, Aarhus, Denmark), 20 g of BSA (Ambion, Cambridgeshire, UK), 3.75 U of SuperTaq DNA polymerase (A. H. Diagnostic, Aarhus, Denmark), and 1 L of DNA solution. The PCR products were checked by electrophoresis on a 2% agarose gel containing 0.1 g of ethidium bromide (BioRad, Hercules, CA)/mL and were viewed by ultraviolet transillumination.
DGGE Ladder A DGGE ladder containing sequences of gastrointestinal bacterial strains was prepared from individual pure cultures (Lactobacillus johnsonii, C. jejuni, E. coli, and Clostridium perfringens). Deoxyribonucleic acid from the pure cultures was extracted using the Qiaamp DNA Mini Kit as specified by the manufacturer (Qiagen, Hilden, Germany), and DNA samples were stored at −20°C. Polymerase chain reaction sequences were obtained from individual pure cultures using the primers HDA1-GC and HDA2, and a PCR program as described previously. The PCR products were mixed in equal amounts to obtain the DGGE ladder.
DGGE Analysis Denaturant gradient gel electrophoresis was performed with a Dcode universal mutation detection system from BioRad using 16-cm × 16-cm × 1-mm gels. The 8% polyacrylamide gels (acrylamide:bisacrylamide = 37.5:1; BioRad) contained a 30 to 55% gradient of urea and formamide (Fluka, Sigma-Aldrich), increasing in the direction of electrophoresis. Electrophoresis was run at 130 V and 60°C in 4 h, after which the gels were stained with SybrGold (1:10,000 dilution; Bie & Berntsen, Roedovre, Denmark) and viewed by ultraviolet transillumination. A total of 6 of 10 samples from each age group of chickens and from both cecal and ileal contents were run on DGGE because of difficulties with the DNA extraction and the PCR amplification of several samples. The intestinal bacterial community profiles of chickens of the same age were compared using the BioNumerics software (Applied Maths BVBA, Sint-Martens-Latem, Belgium). Initially, the DGGE gels were normalized by means of the DGGE markers used, whereafter the software conducted a band search according to a 5% minimum profiling and a 10% gray-zone interval. Subsequently, all bands were checked manually. The manual checking step is essential, but it can cause the loss of objectivity. However, the BioNumerics software is one of the best options for analyzing complex molecular fingerprint data to date (Rademaker et al., 1999). The comparisons were based on the Dice similarity coefficient and the unweighted pair group method using arithmetic means for clustering. Dendrograms reflect
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the grouping and relatedness of samples. The relative similarity between samples can be depicted from the coefficient bar above each diagram in Figures 1 to 4.
Identification of Bacteria by Cloning and Sequencing Samples from 2 chickens of each age group and from each intestinal compartment (ileum and cecum) were cloned using primer pair 26F (5′-AGA GTT TGA TCC TGG CTC A- 3′) and 1390R (5′-GAC GGG CGG TGT GTA CAA3′) (DNA-Technology) and the following amplification program: 1 min at 93°C followed by 25 cycles of 30 s at 92°C, 60 s at 57°C, and 45 s at 72°C. In the last cycle, the 72°C step was extended for 5 min, and the samples were finally cooled down to 4°C. The PCR product was purified using the QIAquick PCR purification kit (Qiagen) and cloned into E. coli using the Topo XL cloning kit (Invitrogen, Carlsbad, CA) as specified by the manufacturer. All clones were checked by DGGE (using primer pair HDA1-GC and HDA2) and selected for sequencing on the basis of their migration in the gel. In total, 120 clones from cecal samples and 90 clones from ileal samples were screened. Plasmid DNA of selected clones was purified using the GenElute Plasmid Miniprep Kit (Sigma-Aldrich). For sequencing of clones, the Dyenamic ET Terminator Cycle Sequencing Kit from Amersham Biosciences and primer 341F (5′-CCC ACG GGA GGC AGC AG-3′; DNATechnology) were used, and the sequencing was carried out on an ABI 3100 capillary DNA analyzing system. The retrieved sequences were compared with the Genbank database using BLAST algorithm (Altschul et al., 1990).
RESULTS Campylobacter Enumeration by Plate Counts No Campylobacter was detected in the transport boxes, the feed and water samples, or the day-old chickens. The results of the Campylobacter counts from the cecal and ileal contents are shown in Table 1. In 2-d-old chickens, C. jejuni was detected in 2 of 10 cecal samples and in 5 of 10 ileal samples (Table 1). The C. jejuni counts within both the cecal and the ileal samples varied by about 3 orders of magnitude. From d 9, all chickens investigated were colonized by C. jejuni. In all infected chickens that were slaughtered at 9 or 17 d of age, larger numbers of C. jejuni were detected by plate counts in both cecum and ileum samples. Generally, numbers of C. jejuni cfu were at least 2 orders of magnitude higher in cecum samples than in ileum samples. In the 9- and 17-d-old chickens, a higher variation in C. jejuni cfu was observed in ileal samples than in cecal samples.
DGGE Profiles and Sequences Obtained from Cecal Content The DGGE profile of the cecal bacterial community of 2-d-old control chickens and chickens infected with C. je-
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juni is shown in Figure 1. The DGGE band patterns of the samples clustered into 2 distinct groups comprising the infected chickens (group 1) and the control chickens (group 2), as is illustrated by the dendrogram. The similarity index between the 2 groups was approximately 63%. The withingroup similarity index of group 1 (infected chickens) was approximately 74%, and the average within-group similarity value of group 2 (control chickens) was approximately 78%. Two of the band patterns obtained from infected chickens and 3 patterns obtained from control chickens were identical. Generally occurring and indicative DGGE bands were sequenced and assigned to a species or a genus in the GenBank database using BLAST (Figures 1 and 2). In the 2-d-old chickens, the sequenced fragments belonged to the genera Enterococcus, Lactobacillus, Clostridium, Weisella, or the species E. coli. 16S rDNA from C. jejuni was found in the clone library from cecal material of infected chickens; however, a faint band identified as C. jejuni 16S rDNA was only obtained from one chicken (chicken 18, Figure 1). In the infected 2d-old chickens, at least 2 distinct bands were absent compared with DGGE gels obtained with material from control chickens (marked in Figure 1, chicken 4). One of these bands could be affiliated to Lactobacillus reuteri, whereas the other band could not be affiliated to 16S rDNA sequences present in the clone library. Generally, the patterns obtained from the infected chickens had a higher within-group similarity index than those from the control chickens. The statistical comparisons of cecal DGGE profiles of 9-d-old (Figure 2) and 17-d-old chickens (data not shown) resulted in 2 major groups as well. However, the fingerprints of two 9-d-old control chickens (Figure 2) and of one 17-d-old control chicken (data not shown) were slightly more similar to the infected chickens than to the control birds. Yet, the band pattern of these control chickens represented the first branch in the groups of infected birds, showing approximately 57 and 47% similarity to the groups of infected chickens, respectively. The comparison of DNA fragments from the 9- and 17d-old chickens with GenBank sequences demonstrated the presence of numerous strains belonging to the genera Clostridium, Lactobacillus, and to the species E. coli. In addition, one fragment, which was only found in the 9-d-old control chickens, could be affiliated to a member of the genus Klebsiella (marked in Figure 2, chicken 1). In total, 16 of 36 sequenced DNA fragments (39%) from cecal material from chickens 2 to 17 d of age could be affiliated to sequences of uncultured bacteria that were related to the genera Clostridium, Ruminococcus, or Lactobacillus.
DGGE Profiles and Sequences Obtained from Ileal Content The DGGE profile of the ileal bacterial community of 2d-old control chickens and chickens infected with C. jejuni is shown in Figure 3. Cluster analysis revealed 2 distinct
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JOHANSEN ET AL. Table 1. Counts of Campylobacter jejuni in cecal and ileal contents of infected chickens1 Age of chicken (d) 2 2 — — — — — — — — — 9 9 — — — — — — — — — 17 17 — — — — — — — — —
Counts (cfu) Group
Chickens (n)
Chicken no.
Cecum
Ileum
Control Infected at 1 d of age — — — — — — — — — Control Infected at 1 d of age — — — — — — — — — Control Infected at 1 d of age — — — — — — — — —
10 10 — — — — — — — — — 10 10 — — — — — — — — — 10 10 — — — — — — — — —
1–10 11 12 13 14 15 16 17 18 19 20 1–10 11 12 13 14 15 16 17 18 19 20 1–10 11 12 13 14 15 16 17 18 19 20
ND2 0 0 0 0 0 0 0 3.6 × 109 0 5.3 × 106 ND 3.1 × 109 6.0 × 108 1.5 × 108 1.7 × 108 5.5 × 108 4.0 × 108 1.4 × 109 1.3 × 109 2.6 × 108 4.1 × 108 ND 5.5 × 108 3.4 × 108 1.3 × 109 6.7 × 109 1.3 × 109 3.7 × 108 1.9 × 109 7.4 × 108 4.4 × 108 1.5 × 109
ND 0 0 5.4 × 107 0 0 0 7.7 × 105 5.9 × 104 9.7 × 104 7.8 × 104 ND 9.3 × 106 4.6 × 106 4.5 × 105 1.4 × 106 1.0 × 106 1.4 × 105 1.8 × 106 7.5 × 106 1.2 × 106 1.6 × 106 ND 1.1 × 107 1.2 × 108 3.0 × 103 1.6 × 105 5.0 × 103 3.5 × 105 2.1 × 104 2.2 × 105 5.1 × 105 6.8 × 104
1
The C. jejuni infection is specified as colony-forming units per gram of intestinal content. ND = no C. jejuni detected in cloacal swab.
2
groups of chickens, consisting of the control chickens and the C. jejuni-infected chickens, respectively. The 2 groups were approximately 33% similar. The infected chickens showed approximately 39% similarity within the group, whereas the within-group similarity of uninfected chickens was approximately 65%. Five distinct fragments of 2-d-old chickens could be affiliated to Enterococcus sp., Lactobacillus ultunensis, L. reuteri, Pediococcus acidilactici, and C. perfringens, respectively. Visual comparison of the DGGE band patterns of control and infected chickens revealed that 3 fragments (marked in Figure 3, chicken 3), including L. reuteri and C. perfringens, which were present in the control chickens, were not detected in the chickens infected with C. jejuni. The third missing fragment was not found among the screened clones. The DGGE profiles and dendrograms generated from the ileal content of 9-d-old (Figure 4) and 17-d-old chickens (data not shown) showed no distinct grouping of the control chickens and the infected chickens. Several fragments could be affiliated to describe microorganisms as illustrated in Figures 3 and 4.
DISCUSSION In this study, the development of bacterial communities in 2 segments of the gastrointestinal tract of broiler chickens
was investigated following an experimental infection with C. jejuni using DGGE on 16S rDNA amplicons. We demonstrated an effect of a C. jejuni infection on the development of the microbial communities of the ceca, but we found no permanent effect of a C. jejuni infection on the ileal microbial community of the chickens. This may be explained by difference in the anatomic structure of the gastrointestinal tract. The cecum is a blind sac with a long retention time of the feed, whereas the ileum is a straight intestinal canal with a much higher flow rate (Savage, 1977). Thus, the environment of the cecum is more stable than the environment of the ileum. The high flow of feed through the ileum may hinder C. jejuni from affecting the microbial community of this compartment. In previous studies (Welkos, 1984; Beery et al., 1988), it has been shown that C. jejuni primarily colonizes the lower gastrointestinal tract, especially the cecum. In agreement with these studies, we observed that the number of C. jejuni recovered from the ceca, with 2 exceptions, was at least 100 times higher than the numbers recovered from the ilea (Table 1). The higher cell numbers of C. jejuni in the ceca may affect the community structure of the infected compartments. Finally, the environment surrounding the chickens may have an influence on the structure of the microbial community of the gastrointestinal tract (Savage, 1977). In our study, the chickens were housed in 2 separate isolators,
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Figure 1. Denaturant gradient gel electrophoresis profile generated from cecal content of 2-d-old chickens using universal primers. M = molecular marker. The chicken number (Chicken #) is denoted above the image, and the group [control chickens (C) and chickens infected at 1 d of age with Campylobacter jejuni (I)] of the chickens is indicated below the image. The relatedness of the band patterns is indicated by their grouping on the dendrogram. Bands identified in control chickens but not in the infected chickens are marked (>). Identified bands are marked (a-i), and the most closely related organisms from GenBank are a) Escherichia coli, b) Lactobacillus reuteri, c) Clostridium disporicum, d) Clostridium paraputrificum, e) Enterococcus sp., f) Weisella sp., g) Enterococcus gallinarum, h) Enterococcus sp., and i) C. jejuni.
where all possible environmental factors were controlled during the entire experiment. Still, it cannot be excluded that different bacterial populations develop randomly in the 2 isolators. However, chickens within the same isolator exhibited unique DGGE fingerprints, suggesting a low environmental impact. Even though we were unable to detect C. jejuni by plate counts (Table 1) in some of the 2-d-old chickens, differences in the DGGE band patterns were already observed between infected and noninfected chickens in both the ileum and
the cecum at that time. Thus, C. jejuni might have affected the development of the microflora before they were detectable by plate counts. The cecal and ileal contents of chickens contained a bacterial community in which enterococci, coliforms, lactobacilli, and clostridia were commonly detected. The DGGE profiles generated from the cecal content revealed several bands that were present in the control chickens but not in the chickens infected with C. jejuni. The microorganisms representing these bands (L. reuteri,
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Figure 2. Denaturant gradient gel electrophoresis profile generated from cecal content of 9-d-old chickens using universal primers. M = molecular marker. The chicken number (Chicken #) is denoted above the image, and the group [control chickens (C) and chickens infected at 1 d of age with Campylobacter jejuni (I)) of the chickens is indicated below the image. The relatedness of the band patterns is indicated by their grouping on the dendrogram. A band identified in the control chickens but not in the infected chickens is marked (>). Identified bands are marked (a-f), and the most closely related organisms from GenBank are a) Clostridium indolis, b) Escherichia coli, c) Klebsiella sp., d) Clostridium sp., e) Lactobacillus ultunensis, and f) Lactobacillus kitasatonis.
Klebsiella sp., C. perfringens) were absent or present in lower numbers in the infected chickens. The bacteria that were suppressed by the infection were first of all identified in the younger chickens. This may partly be due to the visual identification of bands on the DGGE gels. As the complexity of the band patterns increased with age of the chickens (van der Wielen et al.,
2002; Hume et al., 2003), it became more difficult to distinguish single bands unambiguously. Some of the cloned 16S rDNA sequences that were successfully affiliated to known species, including C. jejuni, were not visible on the DGGE gel images. In only one case (Figure 1, chicken 18), a band identified as C. jejuni was observed. In this sample, 109 C. jejuni cfu/g of cecal content
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Figure 3. Denaturant gradient gel electrophoresis profile generated from ileal content of 2-d-old chickens using universal primers. M = molecular marker. The chicken number (Chicken #) is denoted above the image, and the group [control chickens (C) and chickens infected at 1 d of age with Campylobacter jejuni (I)] of the chickens is indicated below the image. The relatedness of the band patterns is indicated by their grouping on the dendrogram. Bands identified in control chickens but not in the infected chickens are marked (>). Identified bands are marked (a-e), and the most related organisms from GenBank are a) Clostridium perfringens, b) Lactobacillus reuteri, c) Lactobacillus ultunensis, d) Pediococcus acidilactici, and e) Enterococcus sp.
was detected by plate count (Table 1), whereas all other cecal samples from the 2-d-old chickens contained lower numbers. This observation is in agreement with results from a study by Simpson et al. (2000), who concluded that the detection limit for cecal bacteria by DGGE (using universal primers) is approximately 108 to 109 cfu/g. This number corresponds to approximately 1% of the microflora in the ceca of chickens (Barnes, 1979). Generally, Muyzer
et al. (1993) estimated that species making up <1% of the community might be overlooked. However, the detection limit of specific groups of bacteria can be improved by using group-specific PCR primers, and it is also possible to obtain more information about the species composition by a subsequent hybridization step using taxon-specific oligonucleotide probes (Muyzer et al., 1993; Muyzer and Ramsing, 1995).
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Figure 4. Denaturant gradient gel electrophoresis profile generated from ileal content of 9-d-old chickens using universal primers. M = molecular marker. The chicken number (Chicken #) is denoted above the image, and the group [control chickens (C) and chickens infected at 1 d of age with C. jejuni (I)] of the chickens is indicated below the image. The relatedness of the band patterns is indicated by their grouping on the dendrogram. Identified bands are marked (a–d), and the most related organisms from GenBank are a) Lactobacillus ultunensis, b) Lactobacillus reuteri, c) Escherichia coli, and d) Clostridium perfringens.
In summary, it is concluded that the DGGE technique is a useful method to investigate changes in the gastrointestinal microflora of chickens, as a consequence of perturbations, such as infections with specific microorganisms. However, to identify actual differences, it is necessary to combine the DGGE method with a hybridization step or
a cloning and sequencing step, as used in this study. Using DGGE, we demonstrated an effect of a C. jejuni infection on the development of the microbial communities of the ceca, but we found no permanent effect of a C. jejuni infection on the ileal microflora of the broiler chickens. In addition, several DGGE bands were detected in the control
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chickens, but were absent in the chickens infected with C. jejuni. Future studies should attempt to investigate the effect of a C. jejuni infection in more detail.
REFERENCES Altschul, S. F., W. Gish, W. Miller, E. W. Myers, and D. J. Lipman. 1990. Basic local alignment search tool. J. Mol. Biol. 215:403– 410. Apajalahti, J. H., A. Kettunen, M. R. Bedford, and W. E. Holben. 2001. Percent G+C profiling accurately reveals diet-related differences in the gastrointestinal microbial community of broiler chickens. Appl. Environ. Microbiol. 67:5656–5667. Barnes, E. M. 1979. The intestinal microflora of poultry and game birds during life and after storage. J. Appl. Bacteriol. 46:407–419. Beery, J. T., M. B. Hugdahl, and M. P. Doyle. 1988. Colonization of gastrointestinal tracts of chicks by Campylobacter jejuni. Appl. Environ. Microbiol. 54:2365–2370. Casamayor, E. O., R. Massana, S. Benlloch, L. Ovreas, B. Diez, V. J. Goddard, J. M. Gasol, I. Joint, F. Rodriguez-Valera, and C. Pedros-Alio. 2002. Changes in archaeal, bacterial and eukaryal assemblages along a salinity gradient by comparison of genetic fingerprinting methods in a multipond solar saltern. Environ. Microbiol. 4:338–348. CDC. 2004. Centers for Disease Control and Prevention, Department of Health and Human Services. http://www.cdc.gov Accessed Nov. 1, 2004. Coates, M. E., R. Fuller, G. F. Harrison, M. Lev, and S. F. Suffolk. 1963. A comparison of the growth of chicks in the Gustafsson germ-free apparatus and in a conventional environment, with and without dietary supplements of penicillin. Br. J. Nutr. 17:141–150. Cocolin, L., M. Manzano, C. Cantoni, and G. Comi. 2001. Denaturing gradient gel electrophoresis analysis of the 16S rRNA gene V1 region to monitor dynamic changes in the bacterial population during fermentation of Italian sausages. Appl. Environ. Microbiol. 67:5113–5121. Coloe, P. J., T. J. Bagust, and L. Ireland. 1984. Development of the normal gastrointestinal microflora of specific pathogenfree chickens. J. Hyg. (Lond.) 92:79–87. Craven, S. E. 2000. Colonization of the intestinal tract by Clostridium perfringens and fecal shedding in diet-stressed and unstressed broiler chickens. Poult. Sci. 79:843–849. Heres, L., B. Engel, F. Van Knapen, J. A. Wagenaar, and B. A. Urlings. 2003. Effect of fermented feed on the susceptibility for Campylobacter jejuni colonisation in broiler chickens with and without concurrent inoculation of Salmonella enteritidis. Int. J. Food Microbiol. 87:75–86. Hume, M. E., L. F. Kubena, T. S. Edrington, C. J. Donskey, R. W. Moore, S. C. Ricke, and D. J. Nisbet. 2003. Poultry digestive microflora biodiversity as indicated by denaturing gradient gel electrophoresis. Poult. Sci. 82:1100–1107. Knarreborg, A., M. A. Simon, R. M. Engberg, B. B. Jensen, and G. W. Tannock. 2002. Effects of dietary fat source and subtherapeutic levels of antibiotic on the bacterial community in the ileum of broiler chickens at various ages. Appl. Environ. Microbiol. 68:5918–5924. Muyzer, G., E. C. de Waal, and A. G. Uitterlinden. 1993. Profiling of complex microbial populations by denaturing gradient gel electrophoresis analysis of polymerase chain reaction-ampli-
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fied genes coding for 16S rRNA. Appl. Environ. Microbiol. 59:695–700. Muyzer, G., and N. B. Ramsing. 1995. Molecular methods to study the organization of microbial communities. Water Sci. Tech. 32:1–9. Netherwood, T., H. J. Gilbert, D. S. Parker, and A. G. O’Donnell. 1999. Probiotics shown to change bacterial community structure in the avian gastrointestinal tract. Appl. Environ. Microbiol. 65:5134–5138. Pearson, A. D., M. H. Greenwood, J. Donaldson, T. D. Healing, D. M. Jones, M. Shahamat, R. K. Feltham, and R. R. Colwell. 2000. Continuous source outbreak of campylobacteriosis traced to chicken. J. Food Prot. 63:309–314. Rademaker, J. L. W., F. J. Louws, U. Rossbach, P. Vinuesa, and F. J. De Bruijn. 1999. Computer-assisted pattern analysis of molecular fingerprints and database construction. Mol. Microb. Ecol. Manual 7.1.3:1–33. Salanitro, J. P., I. G. Blake, and P. A. Muirhead. 1974. Studies on the cecal microflora of commercial broiler chickens. Appl. Microbiol. 28:439–447. Savage, D. C. 1977. Microbial ecology of the gastrointestinal tract. Annu. Rev. Microbiol. 31:107–133. Simpson, J. M., V. J. McCracken, H. R. Gaskins, and R. I. Mackie. 2000. Denaturing gradient gel electrophoresis analysis of 16S ribosomal DNA amplicons to monitor changes in fecal bacterial populations of weaning pigs after introduction of Lactobacillus reuteri strain MM53. Appl. Environ. Microbiol. 66:4705–4714. Stern, N. J., K. L. Hiett, G. A. Alfredsson, K. G. Kristinsson, J. Reiersen, H. Hardardottir, H. Briem, E. Gunnarsson, F. Georgsson, R. Lowman, E. Berndtson, A. M. Lammerding, G. M. Paoli, and M. T. Musgrove. 2003. Campylobacter spp. in Icelandic poultry operations and human disease. Epidemiol. Infect. 130:23–32. Tannock, G. W., K. Munro, H. J. Harmsen, G. W. Welling, J. Smart, and P. K. Gopal. 2000. Analysis of the fecal microflora of human subjects consuming a probiotic product containing Lactobacillus rhamnosus DR20. Appl. Environ. Microbiol. 66:2578–2588. Tauxe, R. V. 1992. Epidemiology of Campylobacter jejuni infections in the United States and other industrialized nations. Am. Soc. Microbiol., Washington, DC. Tauxe, R. V. 2002. Emerging foodborne pathogens. Int. J. Food Microbiol. 78:31–41. Torsvik, V., R. Srheim, and J. Goksoøyr. 1996. Total bacterial diversity in soil and sediment communities—A review. Ind. Microbiol. Biotech. 17:170–178. van der Wielen, P. W., D. A. Keuzenkamp, L. J. Lipman, F. van Knapen, and S. Biesterveld. 2002. Spatial and temporal variation of the intestinal bacterial community in commercially raised broiler chickens during growth. Microb. Ecol. 44:286–293. Vellinga, A., and F. Van Loock. 2002. The dioxin crisis as experiment to determine poultry-related Campylobacter enteritis.Emerg. Infect. Dis. 8:19–22. Walter, J., G. W. Tannock, A. Tilsala-Timisjarvi, S. Rodtong, D. M. Loach, K. Munro, and T. Alatossava. 2000. Detection and identification of gastrointestinal Lactobacillus species by using denaturing gradient gel electrophoresis and species-specific PCR primers. Appl. Environ. Microbiol. 66:297–303. Welkos, S. L. 1984. Experimental gastroenteritis in newly-hatched chicks infected with Campylobacter jejuni. J. Med. Microbiol. 18:233–248.
ABSTRACT The effect of a Campylobacter jejuni colonization on the development of the microflora of the cecum and the ileum of broiler chickens was studied using molecular methods. The infection did affect the development and complexity of the microbial communities of the ceca, but we found no permanent effect of a C. jejuni infection on the ileal microflora of the broilers. In addition, denatur-
Key words: broiler chicken, Campylobacter, gut microflora, denaturant gradient gel electrophoresis 2006 Poultry Science 85:579–587
fact that far from all bacteria are culturable in vitro, which has been discovered after the introduction of culture-independent molecular methods in microbial ecology (Torsvik et al., 1996; Tannock et al., 2000). Over the last decade, denaturant gradient gel electrophoresis (DGGE; Muyzer et al., 1993) has been used successfully to investigate the diversity of complex bacterial communities. This method has been widely used to monitor temporal and spacial changes in community structure in response to changes in environmental parameters (Simpson et al., 2000; Cocolin et al., 2001; Casamayor et al., 2002). The aim of the present study was to examine and identify differences in the development of the bacterial community of the ceca and ilea of 2-, 9- and 17-d-old broiler chickens experimentally infected at 1 d of age with C. jejuni compared with uninfected control chickens. This was done by comparing DGGE banding patterns from the different groups of chickens.
INTRODUCTION Campylobacter jejuni, a natural inhabitant of the gastrointestinal tract of chickens and other birds, is currently the most common cause of acute bacterial gastroenteritis (Tauxe, 1992, 2002; CDC, 2004). Increasing evidence suggests a strong association between poultry and human illness caused by C. jejuni (Pearson et al., 2000; Vellinga and Van Loock, 2002; Stern et al., 2003). Levels of Campylobacter in the cecum of infected chickens tend to be high, and typically reported cfu values are 108 to 109/g of content (Heres et al., 2003). Preventing contamination of poultry and poultry products with foodborne human pathogens such as Campylobacter is a challenge for commercial producers worldwide. It is well recognized that the gastrointestinal microbial community of broiler chickens plays an important role in the growth and health of the birds and that the intestinal bacterial composition can be affected by various factors such as diet, stress, probiotics, and antibiotics (Coates et al., 1963; Netherwood et al., 1999; Craven, 2000; Apajalahti et al., 2001; Knarreborg et al., 2002). Generally, little is known about the composition and dynamics of the intestinal microbial community of chickens. Studies on the composition of the intestinal microflora have traditionally been carried out using classical microbiological culture methods (Salanitro et al., 1974; Barnes, 1979; Coloe et al., 1984). However, culture-based diversity studies are biased by the
MATERIALS AND METHODS Animals, Experimental Infection, and Sampling The study was carried out with 60 conventional broiler chickens (Ross 208) purchased at 1 d of age from a local hatchery (Fællesrugeriet, Randers, Denmark). The chickens were transferred directly from the hatchery to the experimental unit, where they were housed in isolators (HM 1500, Montair Andersen B.V., Sevenum, The Netherlands). Prior to the experimental infection, transport boxes and feed and water samples from each isolator were analyzed for the presence of Campylobacter by cultivation. The day-
2006 Poultry Science Association, Inc. Received September 28, 2005. Accepted December 19, 2005. 1 Corresponding author: [email protected]
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old chickens were checked for Campylobacter by cloacal swabs. One group of 30, 1-d-old chickens was experimentally infected orally with 6 × 106 cfu of C. jejuni strain DVI-sc181. The control group of 30 chickens was given a 0.9% NaCl solution. The chickens had access to feed and water ad libitum. They were fed conventional broiler feed without antimicrobial additives (Danish Institute of Agricultural Sciences, Foulum, Denmark). From each isolator, 10 birds were randomly selected at ages 2, 9, and 17 d and killed by decapitation. The number of C. jejuni (cfu) in the cecal and the ileal contents of the chickens was quantified by the spread-plate method on modified charcoal cefoperazone deoxycholate agar (Oxoid, Basingstoke, UK). In addition, samples from the ceca and ilea were collected in Eppendorf tubes for a 16S rDNA-based study of the microbial community and stored at −80°C until further processing.
DNA Extraction Deoxyribonucleic acid was extracted from the cecal and the ileal contents of broilers. For DNA extraction, 250 mg of intestinal content was suspended in 600 L of PBS. The suspension was mixed thoroughly and centrifuged at 200 × g for 2 min. The supernatant was transferred to a new Eppendorf tube and centrifuged at 12,000 × g for 5 min. Subsequently, the pellet was resuspended in 570 L of TEbuffer (10 mM Tris, 1 mM EDTA; pH 8.0) and transferred to a screw-cap tube containing 400 g of sterile zirconium beads (diameter = 0.1 mm; Biospec Products, Bartlesville, OK). The cells were lysed by physical disruption for 3.5 min in a minibead beater (Techtum, Umea˚, Sweden) on high speed. After homogenization, the sample was centrifuged, and the supernatant was transferred to a new tube to which 100 L of 5 M NaCl and 80 L of CTAB/NaCl [10% CTAB (wt/vol) = hexadecyltrimethyl ammonium bromide; Sigma-Aldrich, St. Louis, MO), 0.7 M NaCl) was added. The suspension was mixed thoroughly and placed in a water bath for 10 min at 65°C. The sample was extracted 3 times with 0.7 mL of chloroform:isoamyl alcohol (24:1), 0.7 mL of phenol:chloroform:isoamyl alcohol (25:24:1), and 0.6 mL of chloroform:isoamyl alcohol (24:1), respectively (Sigma-Aldrich). The DNA was precipitated with 99.9% cold ethanol. Finally, the DNA was dissolved in 80 L of TE-buffer and stored at −20°C until further use.
PCR Amplification with Universal 16S rDNA Primers Polymerase chain reaction amplifications of total bacterial community DNA were performed using primer pair HDA1-GC (5′-CGC CCG GGG CGC GCC CCG GGC GGG GCG GGG GCA CGG GGG GAC TCC TAC GGG AGG CAG CAG T-3′; the GC-clamp is in boldface) and HDA2 (5′-GTA TTA CCG CGG CTG CTG GCA C-3′; Walter et al., 2000). These primers amplify the V2-V3 region of the 16S rDNA gene (position 339-539 in Escherichia coli), resulting in fragments of 200 bp. The following thermocycling program was used: 94°C for 4.5 min; 30 cycles of
94°C for 30 s, 56°C for 30 s, and 68°C for 1 min; and finally, 68°C for 7 min (Walter et al., 2000). Polymerase chain reaction was performed in 0.2-mL tubes using a Peltier Thermal Cycler 200 (MJ Research Inc., Watertown, MA). The reaction mixture (50 L) contained 1 × SuperTaq reaction buffer, 200 M of each dNTP (Amersham Biosciences, Piscataway, NJ), 20 pmol of each primer (DNA-Technology, Aarhus, Denmark), 20 g of BSA (Ambion, Cambridgeshire, UK), 3.75 U of SuperTaq DNA polymerase (A. H. Diagnostic, Aarhus, Denmark), and 1 L of DNA solution. The PCR products were checked by electrophoresis on a 2% agarose gel containing 0.1 g of ethidium bromide (BioRad, Hercules, CA)/mL and were viewed by ultraviolet transillumination.
DGGE Ladder A DGGE ladder containing sequences of gastrointestinal bacterial strains was prepared from individual pure cultures (Lactobacillus johnsonii, C. jejuni, E. coli, and Clostridium perfringens). Deoxyribonucleic acid from the pure cultures was extracted using the Qiaamp DNA Mini Kit as specified by the manufacturer (Qiagen, Hilden, Germany), and DNA samples were stored at −20°C. Polymerase chain reaction sequences were obtained from individual pure cultures using the primers HDA1-GC and HDA2, and a PCR program as described previously. The PCR products were mixed in equal amounts to obtain the DGGE ladder.
DGGE Analysis Denaturant gradient gel electrophoresis was performed with a Dcode universal mutation detection system from BioRad using 16-cm × 16-cm × 1-mm gels. The 8% polyacrylamide gels (acrylamide:bisacrylamide = 37.5:1; BioRad) contained a 30 to 55% gradient of urea and formamide (Fluka, Sigma-Aldrich), increasing in the direction of electrophoresis. Electrophoresis was run at 130 V and 60°C in 4 h, after which the gels were stained with SybrGold (1:10,000 dilution; Bie & Berntsen, Roedovre, Denmark) and viewed by ultraviolet transillumination. A total of 6 of 10 samples from each age group of chickens and from both cecal and ileal contents were run on DGGE because of difficulties with the DNA extraction and the PCR amplification of several samples. The intestinal bacterial community profiles of chickens of the same age were compared using the BioNumerics software (Applied Maths BVBA, Sint-Martens-Latem, Belgium). Initially, the DGGE gels were normalized by means of the DGGE markers used, whereafter the software conducted a band search according to a 5% minimum profiling and a 10% gray-zone interval. Subsequently, all bands were checked manually. The manual checking step is essential, but it can cause the loss of objectivity. However, the BioNumerics software is one of the best options for analyzing complex molecular fingerprint data to date (Rademaker et al., 1999). The comparisons were based on the Dice similarity coefficient and the unweighted pair group method using arithmetic means for clustering. Dendrograms reflect
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the grouping and relatedness of samples. The relative similarity between samples can be depicted from the coefficient bar above each diagram in Figures 1 to 4.
Identification of Bacteria by Cloning and Sequencing Samples from 2 chickens of each age group and from each intestinal compartment (ileum and cecum) were cloned using primer pair 26F (5′-AGA GTT TGA TCC TGG CTC A- 3′) and 1390R (5′-GAC GGG CGG TGT GTA CAA3′) (DNA-Technology) and the following amplification program: 1 min at 93°C followed by 25 cycles of 30 s at 92°C, 60 s at 57°C, and 45 s at 72°C. In the last cycle, the 72°C step was extended for 5 min, and the samples were finally cooled down to 4°C. The PCR product was purified using the QIAquick PCR purification kit (Qiagen) and cloned into E. coli using the Topo XL cloning kit (Invitrogen, Carlsbad, CA) as specified by the manufacturer. All clones were checked by DGGE (using primer pair HDA1-GC and HDA2) and selected for sequencing on the basis of their migration in the gel. In total, 120 clones from cecal samples and 90 clones from ileal samples were screened. Plasmid DNA of selected clones was purified using the GenElute Plasmid Miniprep Kit (Sigma-Aldrich). For sequencing of clones, the Dyenamic ET Terminator Cycle Sequencing Kit from Amersham Biosciences and primer 341F (5′-CCC ACG GGA GGC AGC AG-3′; DNATechnology) were used, and the sequencing was carried out on an ABI 3100 capillary DNA analyzing system. The retrieved sequences were compared with the Genbank database using BLAST algorithm (Altschul et al., 1990).
RESULTS Campylobacter Enumeration by Plate Counts No Campylobacter was detected in the transport boxes, the feed and water samples, or the day-old chickens. The results of the Campylobacter counts from the cecal and ileal contents are shown in Table 1. In 2-d-old chickens, C. jejuni was detected in 2 of 10 cecal samples and in 5 of 10 ileal samples (Table 1). The C. jejuni counts within both the cecal and the ileal samples varied by about 3 orders of magnitude. From d 9, all chickens investigated were colonized by C. jejuni. In all infected chickens that were slaughtered at 9 or 17 d of age, larger numbers of C. jejuni were detected by plate counts in both cecum and ileum samples. Generally, numbers of C. jejuni cfu were at least 2 orders of magnitude higher in cecum samples than in ileum samples. In the 9- and 17-d-old chickens, a higher variation in C. jejuni cfu was observed in ileal samples than in cecal samples.
DGGE Profiles and Sequences Obtained from Cecal Content The DGGE profile of the cecal bacterial community of 2-d-old control chickens and chickens infected with C. je-
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juni is shown in Figure 1. The DGGE band patterns of the samples clustered into 2 distinct groups comprising the infected chickens (group 1) and the control chickens (group 2), as is illustrated by the dendrogram. The similarity index between the 2 groups was approximately 63%. The withingroup similarity index of group 1 (infected chickens) was approximately 74%, and the average within-group similarity value of group 2 (control chickens) was approximately 78%. Two of the band patterns obtained from infected chickens and 3 patterns obtained from control chickens were identical. Generally occurring and indicative DGGE bands were sequenced and assigned to a species or a genus in the GenBank database using BLAST (Figures 1 and 2). In the 2-d-old chickens, the sequenced fragments belonged to the genera Enterococcus, Lactobacillus, Clostridium, Weisella, or the species E. coli. 16S rDNA from C. jejuni was found in the clone library from cecal material of infected chickens; however, a faint band identified as C. jejuni 16S rDNA was only obtained from one chicken (chicken 18, Figure 1). In the infected 2d-old chickens, at least 2 distinct bands were absent compared with DGGE gels obtained with material from control chickens (marked in Figure 1, chicken 4). One of these bands could be affiliated to Lactobacillus reuteri, whereas the other band could not be affiliated to 16S rDNA sequences present in the clone library. Generally, the patterns obtained from the infected chickens had a higher within-group similarity index than those from the control chickens. The statistical comparisons of cecal DGGE profiles of 9-d-old (Figure 2) and 17-d-old chickens (data not shown) resulted in 2 major groups as well. However, the fingerprints of two 9-d-old control chickens (Figure 2) and of one 17-d-old control chicken (data not shown) were slightly more similar to the infected chickens than to the control birds. Yet, the band pattern of these control chickens represented the first branch in the groups of infected birds, showing approximately 57 and 47% similarity to the groups of infected chickens, respectively. The comparison of DNA fragments from the 9- and 17d-old chickens with GenBank sequences demonstrated the presence of numerous strains belonging to the genera Clostridium, Lactobacillus, and to the species E. coli. In addition, one fragment, which was only found in the 9-d-old control chickens, could be affiliated to a member of the genus Klebsiella (marked in Figure 2, chicken 1). In total, 16 of 36 sequenced DNA fragments (39%) from cecal material from chickens 2 to 17 d of age could be affiliated to sequences of uncultured bacteria that were related to the genera Clostridium, Ruminococcus, or Lactobacillus.
DGGE Profiles and Sequences Obtained from Ileal Content The DGGE profile of the ileal bacterial community of 2d-old control chickens and chickens infected with C. jejuni is shown in Figure 3. Cluster analysis revealed 2 distinct
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JOHANSEN ET AL. Table 1. Counts of Campylobacter jejuni in cecal and ileal contents of infected chickens1 Age of chicken (d) 2 2 — — — — — — — — — 9 9 — — — — — — — — — 17 17 — — — — — — — — —
Counts (cfu) Group
Chickens (n)
Chicken no.
Cecum
Ileum
Control Infected at 1 d of age — — — — — — — — — Control Infected at 1 d of age — — — — — — — — — Control Infected at 1 d of age — — — — — — — — —
10 10 — — — — — — — — — 10 10 — — — — — — — — — 10 10 — — — — — — — — —
1–10 11 12 13 14 15 16 17 18 19 20 1–10 11 12 13 14 15 16 17 18 19 20 1–10 11 12 13 14 15 16 17 18 19 20
ND2 0 0 0 0 0 0 0 3.6 × 109 0 5.3 × 106 ND 3.1 × 109 6.0 × 108 1.5 × 108 1.7 × 108 5.5 × 108 4.0 × 108 1.4 × 109 1.3 × 109 2.6 × 108 4.1 × 108 ND 5.5 × 108 3.4 × 108 1.3 × 109 6.7 × 109 1.3 × 109 3.7 × 108 1.9 × 109 7.4 × 108 4.4 × 108 1.5 × 109
ND 0 0 5.4 × 107 0 0 0 7.7 × 105 5.9 × 104 9.7 × 104 7.8 × 104 ND 9.3 × 106 4.6 × 106 4.5 × 105 1.4 × 106 1.0 × 106 1.4 × 105 1.8 × 106 7.5 × 106 1.2 × 106 1.6 × 106 ND 1.1 × 107 1.2 × 108 3.0 × 103 1.6 × 105 5.0 × 103 3.5 × 105 2.1 × 104 2.2 × 105 5.1 × 105 6.8 × 104
1
The C. jejuni infection is specified as colony-forming units per gram of intestinal content. ND = no C. jejuni detected in cloacal swab.
2
groups of chickens, consisting of the control chickens and the C. jejuni-infected chickens, respectively. The 2 groups were approximately 33% similar. The infected chickens showed approximately 39% similarity within the group, whereas the within-group similarity of uninfected chickens was approximately 65%. Five distinct fragments of 2-d-old chickens could be affiliated to Enterococcus sp., Lactobacillus ultunensis, L. reuteri, Pediococcus acidilactici, and C. perfringens, respectively. Visual comparison of the DGGE band patterns of control and infected chickens revealed that 3 fragments (marked in Figure 3, chicken 3), including L. reuteri and C. perfringens, which were present in the control chickens, were not detected in the chickens infected with C. jejuni. The third missing fragment was not found among the screened clones. The DGGE profiles and dendrograms generated from the ileal content of 9-d-old (Figure 4) and 17-d-old chickens (data not shown) showed no distinct grouping of the control chickens and the infected chickens. Several fragments could be affiliated to describe microorganisms as illustrated in Figures 3 and 4.
DISCUSSION In this study, the development of bacterial communities in 2 segments of the gastrointestinal tract of broiler chickens
was investigated following an experimental infection with C. jejuni using DGGE on 16S rDNA amplicons. We demonstrated an effect of a C. jejuni infection on the development of the microbial communities of the ceca, but we found no permanent effect of a C. jejuni infection on the ileal microbial community of the chickens. This may be explained by difference in the anatomic structure of the gastrointestinal tract. The cecum is a blind sac with a long retention time of the feed, whereas the ileum is a straight intestinal canal with a much higher flow rate (Savage, 1977). Thus, the environment of the cecum is more stable than the environment of the ileum. The high flow of feed through the ileum may hinder C. jejuni from affecting the microbial community of this compartment. In previous studies (Welkos, 1984; Beery et al., 1988), it has been shown that C. jejuni primarily colonizes the lower gastrointestinal tract, especially the cecum. In agreement with these studies, we observed that the number of C. jejuni recovered from the ceca, with 2 exceptions, was at least 100 times higher than the numbers recovered from the ilea (Table 1). The higher cell numbers of C. jejuni in the ceca may affect the community structure of the infected compartments. Finally, the environment surrounding the chickens may have an influence on the structure of the microbial community of the gastrointestinal tract (Savage, 1977). In our study, the chickens were housed in 2 separate isolators,
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Figure 1. Denaturant gradient gel electrophoresis profile generated from cecal content of 2-d-old chickens using universal primers. M = molecular marker. The chicken number (Chicken #) is denoted above the image, and the group [control chickens (C) and chickens infected at 1 d of age with Campylobacter jejuni (I)] of the chickens is indicated below the image. The relatedness of the band patterns is indicated by their grouping on the dendrogram. Bands identified in control chickens but not in the infected chickens are marked (>). Identified bands are marked (a-i), and the most closely related organisms from GenBank are a) Escherichia coli, b) Lactobacillus reuteri, c) Clostridium disporicum, d) Clostridium paraputrificum, e) Enterococcus sp., f) Weisella sp., g) Enterococcus gallinarum, h) Enterococcus sp., and i) C. jejuni.
where all possible environmental factors were controlled during the entire experiment. Still, it cannot be excluded that different bacterial populations develop randomly in the 2 isolators. However, chickens within the same isolator exhibited unique DGGE fingerprints, suggesting a low environmental impact. Even though we were unable to detect C. jejuni by plate counts (Table 1) in some of the 2-d-old chickens, differences in the DGGE band patterns were already observed between infected and noninfected chickens in both the ileum and
the cecum at that time. Thus, C. jejuni might have affected the development of the microflora before they were detectable by plate counts. The cecal and ileal contents of chickens contained a bacterial community in which enterococci, coliforms, lactobacilli, and clostridia were commonly detected. The DGGE profiles generated from the cecal content revealed several bands that were present in the control chickens but not in the chickens infected with C. jejuni. The microorganisms representing these bands (L. reuteri,
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Figure 2. Denaturant gradient gel electrophoresis profile generated from cecal content of 9-d-old chickens using universal primers. M = molecular marker. The chicken number (Chicken #) is denoted above the image, and the group [control chickens (C) and chickens infected at 1 d of age with Campylobacter jejuni (I)) of the chickens is indicated below the image. The relatedness of the band patterns is indicated by their grouping on the dendrogram. A band identified in the control chickens but not in the infected chickens is marked (>). Identified bands are marked (a-f), and the most closely related organisms from GenBank are a) Clostridium indolis, b) Escherichia coli, c) Klebsiella sp., d) Clostridium sp., e) Lactobacillus ultunensis, and f) Lactobacillus kitasatonis.
Klebsiella sp., C. perfringens) were absent or present in lower numbers in the infected chickens. The bacteria that were suppressed by the infection were first of all identified in the younger chickens. This may partly be due to the visual identification of bands on the DGGE gels. As the complexity of the band patterns increased with age of the chickens (van der Wielen et al.,
2002; Hume et al., 2003), it became more difficult to distinguish single bands unambiguously. Some of the cloned 16S rDNA sequences that were successfully affiliated to known species, including C. jejuni, were not visible on the DGGE gel images. In only one case (Figure 1, chicken 18), a band identified as C. jejuni was observed. In this sample, 109 C. jejuni cfu/g of cecal content
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Figure 3. Denaturant gradient gel electrophoresis profile generated from ileal content of 2-d-old chickens using universal primers. M = molecular marker. The chicken number (Chicken #) is denoted above the image, and the group [control chickens (C) and chickens infected at 1 d of age with Campylobacter jejuni (I)] of the chickens is indicated below the image. The relatedness of the band patterns is indicated by their grouping on the dendrogram. Bands identified in control chickens but not in the infected chickens are marked (>). Identified bands are marked (a-e), and the most related organisms from GenBank are a) Clostridium perfringens, b) Lactobacillus reuteri, c) Lactobacillus ultunensis, d) Pediococcus acidilactici, and e) Enterococcus sp.
was detected by plate count (Table 1), whereas all other cecal samples from the 2-d-old chickens contained lower numbers. This observation is in agreement with results from a study by Simpson et al. (2000), who concluded that the detection limit for cecal bacteria by DGGE (using universal primers) is approximately 108 to 109 cfu/g. This number corresponds to approximately 1% of the microflora in the ceca of chickens (Barnes, 1979). Generally, Muyzer
et al. (1993) estimated that species making up <1% of the community might be overlooked. However, the detection limit of specific groups of bacteria can be improved by using group-specific PCR primers, and it is also possible to obtain more information about the species composition by a subsequent hybridization step using taxon-specific oligonucleotide probes (Muyzer et al., 1993; Muyzer and Ramsing, 1995).
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Figure 4. Denaturant gradient gel electrophoresis profile generated from ileal content of 9-d-old chickens using universal primers. M = molecular marker. The chicken number (Chicken #) is denoted above the image, and the group [control chickens (C) and chickens infected at 1 d of age with C. jejuni (I)] of the chickens is indicated below the image. The relatedness of the band patterns is indicated by their grouping on the dendrogram. Identified bands are marked (a–d), and the most related organisms from GenBank are a) Lactobacillus ultunensis, b) Lactobacillus reuteri, c) Escherichia coli, and d) Clostridium perfringens.
In summary, it is concluded that the DGGE technique is a useful method to investigate changes in the gastrointestinal microflora of chickens, as a consequence of perturbations, such as infections with specific microorganisms. However, to identify actual differences, it is necessary to combine the DGGE method with a hybridization step or
a cloning and sequencing step, as used in this study. Using DGGE, we demonstrated an effect of a C. jejuni infection on the development of the microbial communities of the ceca, but we found no permanent effect of a C. jejuni infection on the ileal microflora of the broiler chickens. In addition, several DGGE bands were detected in the control
CAMPYLOBACTER AND THE GUT MICROFLORA
chickens, but were absent in the chickens infected with C. jejuni. Future studies should attempt to investigate the effect of a C. jejuni infection in more detail.
REFERENCES Altschul, S. F., W. Gish, W. Miller, E. W. Myers, and D. J. Lipman. 1990. Basic local alignment search tool. J. Mol. Biol. 215:403– 410. Apajalahti, J. H., A. Kettunen, M. R. Bedford, and W. E. Holben. 2001. Percent G+C profiling accurately reveals diet-related differences in the gastrointestinal microbial community of broiler chickens. Appl. Environ. Microbiol. 67:5656–5667. Barnes, E. M. 1979. The intestinal microflora of poultry and game birds during life and after storage. J. Appl. Bacteriol. 46:407–419. Beery, J. T., M. B. Hugdahl, and M. P. Doyle. 1988. Colonization of gastrointestinal tracts of chicks by Campylobacter jejuni. Appl. Environ. Microbiol. 54:2365–2370. Casamayor, E. O., R. Massana, S. Benlloch, L. Ovreas, B. Diez, V. J. Goddard, J. M. Gasol, I. Joint, F. Rodriguez-Valera, and C. Pedros-Alio. 2002. Changes in archaeal, bacterial and eukaryal assemblages along a salinity gradient by comparison of genetic fingerprinting methods in a multipond solar saltern. Environ. Microbiol. 4:338–348. CDC. 2004. Centers for Disease Control and Prevention, Department of Health and Human Services. http://www.cdc.gov Accessed Nov. 1, 2004. Coates, M. E., R. Fuller, G. F. Harrison, M. Lev, and S. F. Suffolk. 1963. A comparison of the growth of chicks in the Gustafsson germ-free apparatus and in a conventional environment, with and without dietary supplements of penicillin. Br. J. Nutr. 17:141–150. Cocolin, L., M. Manzano, C. Cantoni, and G. Comi. 2001. Denaturing gradient gel electrophoresis analysis of the 16S rRNA gene V1 region to monitor dynamic changes in the bacterial population during fermentation of Italian sausages. Appl. Environ. Microbiol. 67:5113–5121. Coloe, P. J., T. J. Bagust, and L. Ireland. 1984. Development of the normal gastrointestinal microflora of specific pathogenfree chickens. J. Hyg. (Lond.) 92:79–87. Craven, S. E. 2000. Colonization of the intestinal tract by Clostridium perfringens and fecal shedding in diet-stressed and unstressed broiler chickens. Poult. Sci. 79:843–849. Heres, L., B. Engel, F. Van Knapen, J. A. Wagenaar, and B. A. Urlings. 2003. Effect of fermented feed on the susceptibility for Campylobacter jejuni colonisation in broiler chickens with and without concurrent inoculation of Salmonella enteritidis. Int. J. Food Microbiol. 87:75–86. Hume, M. E., L. F. Kubena, T. S. Edrington, C. J. Donskey, R. W. Moore, S. C. Ricke, and D. J. Nisbet. 2003. Poultry digestive microflora biodiversity as indicated by denaturing gradient gel electrophoresis. Poult. Sci. 82:1100–1107. Knarreborg, A., M. A. Simon, R. M. Engberg, B. B. Jensen, and G. W. Tannock. 2002. Effects of dietary fat source and subtherapeutic levels of antibiotic on the bacterial community in the ileum of broiler chickens at various ages. Appl. Environ. Microbiol. 68:5918–5924. Muyzer, G., E. C. de Waal, and A. G. Uitterlinden. 1993. Profiling of complex microbial populations by denaturing gradient gel electrophoresis analysis of polymerase chain reaction-ampli-
587
fied genes coding for 16S rRNA. Appl. Environ. Microbiol. 59:695–700. Muyzer, G., and N. B. Ramsing. 1995. Molecular methods to study the organization of microbial communities. Water Sci. Tech. 32:1–9. Netherwood, T., H. J. Gilbert, D. S. Parker, and A. G. O’Donnell. 1999. Probiotics shown to change bacterial community structure in the avian gastrointestinal tract. Appl. Environ. Microbiol. 65:5134–5138. Pearson, A. D., M. H. Greenwood, J. Donaldson, T. D. Healing, D. M. Jones, M. Shahamat, R. K. Feltham, and R. R. Colwell. 2000. Continuous source outbreak of campylobacteriosis traced to chicken. J. Food Prot. 63:309–314. Rademaker, J. L. W., F. J. Louws, U. Rossbach, P. Vinuesa, and F. J. De Bruijn. 1999. Computer-assisted pattern analysis of molecular fingerprints and database construction. Mol. Microb. Ecol. Manual 7.1.3:1–33. Salanitro, J. P., I. G. Blake, and P. A. Muirhead. 1974. Studies on the cecal microflora of commercial broiler chickens. Appl. Microbiol. 28:439–447. Savage, D. C. 1977. Microbial ecology of the gastrointestinal tract. Annu. Rev. Microbiol. 31:107–133. Simpson, J. M., V. J. McCracken, H. R. Gaskins, and R. I. Mackie. 2000. Denaturing gradient gel electrophoresis analysis of 16S ribosomal DNA amplicons to monitor changes in fecal bacterial populations of weaning pigs after introduction of Lactobacillus reuteri strain MM53. Appl. Environ. Microbiol. 66:4705–4714. Stern, N. J., K. L. Hiett, G. A. Alfredsson, K. G. Kristinsson, J. Reiersen, H. Hardardottir, H. Briem, E. Gunnarsson, F. Georgsson, R. Lowman, E. Berndtson, A. M. Lammerding, G. M. Paoli, and M. T. Musgrove. 2003. Campylobacter spp. in Icelandic poultry operations and human disease. Epidemiol. Infect. 130:23–32. Tannock, G. W., K. Munro, H. J. Harmsen, G. W. Welling, J. Smart, and P. K. Gopal. 2000. Analysis of the fecal microflora of human subjects consuming a probiotic product containing Lactobacillus rhamnosus DR20. Appl. Environ. Microbiol. 66:2578–2588. Tauxe, R. V. 1992. Epidemiology of Campylobacter jejuni infections in the United States and other industrialized nations. Am. Soc. Microbiol., Washington, DC. Tauxe, R. V. 2002. Emerging foodborne pathogens. Int. J. Food Microbiol. 78:31–41. Torsvik, V., R. Srheim, and J. Goksoøyr. 1996. Total bacterial diversity in soil and sediment communities—A review. Ind. Microbiol. Biotech. 17:170–178. van der Wielen, P. W., D. A. Keuzenkamp, L. J. Lipman, F. van Knapen, and S. Biesterveld. 2002. Spatial and temporal variation of the intestinal bacterial community in commercially raised broiler chickens during growth. Microb. Ecol. 44:286–293. Vellinga, A., and F. Van Loock. 2002. The dioxin crisis as experiment to determine poultry-related Campylobacter enteritis.Emerg. Infect. Dis. 8:19–22. Walter, J., G. W. Tannock, A. Tilsala-Timisjarvi, S. Rodtong, D. M. Loach, K. Munro, and T. Alatossava. 2000. Detection and identification of gastrointestinal Lactobacillus species by using denaturing gradient gel electrophoresis and species-specific PCR primers. Appl. Environ. Microbiol. 66:297–303. Welkos, S. L. 1984. Experimental gastroenteritis in newly-hatched chicks infected with Campylobacter jejuni. J. Med. Microbiol. 18:233–248.