Identification of the Most Abundant Lactobacillus Species in the Crop of 1- and 5-Week-Old Broiler Chickens
ABSTRACT
Bacteria from crops of 1- and 5-week-old broiler chickens fed with two brands (diets A and B) of wheat-based diets were isolated on Lactobacillus-selective medium and identified (n = 300) based on partial 16S rRNA gene sequence. The most abundant Lactobacillus species were L. reuteri (33%), L. crispatus (18.7%), and L. salivarius (13.3%). Regardless of farm and feed, L. reuteri was the most abundant species (P < 0.005) in the crops of the younger chickens. However, the amount of L. reuteri was significantly reduced in the crops of the 5-week-old chickens regardless of the feed (P = 0.016). The diversity of L. reuteri isolates was studied by fatty acid analysis, and the 94 L. reuteri isolates could be arranged into several clusters. The nisin sensitivities of the L. reuteri isolates were determined because nisin is a candidate coccidiostat. Sensitive isolates were found more frequently in younger chickens (77%) than in 5-week-old chickens (23%), whereas chickens fed with commercial feed B had a higher proportion of nisin-resistant isolates (73%) than did chickens fed with feed A (45%). Nisin-resistant strains are potential candidates for adjunct cultures for maintaining L. reuteri in its natural niche in the crop and are potential targets for genetic engineering with nisin-selectable food-grade vectors. The diversity of the L. reuteri population suggested that one should consider including several strains representing different clusters and nisin resistance phenotypes in candidate probiotic feed supplements for chickens.
The bacterial colonization of the chicken digestive tract starts from the first hours of life, and each region of the intestinal tract is colonized by a typical microbiota as soon as feed is given (3, 31, 38). Lactic acid bacteria account for an important part of the intestinal microbiota of chickens and contribute to maintaining the ecological balance of the different microorganisms (35, 41, 42). It is commonly agreed that Lactobacillus spp. dominate the anterior small intestine, crop, duodenal and jejunal epithelial cells, and digesta of chicken (47). Early studies on chicken microbiota found that Lactobacillus salivarius, L. reuteri, and L. acidophilus inhabited the crop and that these species were present throughout the chicken digestive tract (31, 35, 38). However, the chicken gastrointestinal microbiota was characterized mainly by culture-based methods, which gave less reliable species identification (1, 2). In addition, most of those studies were done prior to the reclassification of Lactobacillus acidophilus, which has now been divided into two DNA homology groups containing six related species (19, 27). DNA homology group A contains L. acidophilus (A1), L. crispatus (A2), L. amylovorus (A3), and L. gallinarum (A4), whereas DNA homology group B contains L. gasseri (B1) and L. johnsonii (B2). Recently, molecular methods such as 16S RNA gene sequencing and denaturing gradient gel electrophoresis have been used for reliable species identification and for analysis of noncultivable members of the bacterial community of chickens (43).
Lactobacilli efficiently colonize the stratified squamous epithelium lining of the crop, which functions as a food storage pouch in the middle of the esophagus. In the crop, carbohydrate digestion begins with moistening and microbial fermentation (6, 23). The fermenting lactobacilli in the crop secrete acids and lower the pH of crop to pH 4.5 to 6 (35). Lowering the pH by organic acids may improve nutrient absorption (5), showing one beneficial effect of the lactobacilli in the crop. Furthermore, Jin et al. (26) previously reported that all their Lactobacillus strains isolated from the chicken intestine inhibited pathogenic bacteria such as Escherichia coli and Salmonella spp. by the production of organic acids and not via hydrogen peroxide or bacteriocin production. However, Fuller (20) pointed out that the bacteriostatic property of lactobacilli in crop is due to low pH, but the bactericidal activity could not be accounted for by the pH alone. Part of the antimicrobial activity in the crop may arise from small inhibitory organic compounds such as reuterin and antibiotics like reutericyclin, antagonizing gram-negative and gram-positive bacteria, yeast, fungi, protozoa, and viruses (4, 25). L. reuteri, a species which has frequently been isolated from the intestines of humans and animals (10), is capable of producing such antimicrobial compounds. Bacteriocins may play a positive role in the inhibition of pathogens in the crop. For example, chickens eating a diet containing bacteriocin OR-7 had lower Campylobacter jejuni counts than chickens eating the same diet without the bacteriocin (39).
The composition of the chicken bacterial community can be affected by several factors such as diet (29, 37), age (48), antibiotic administration (29), and infection with pathogens (28). There is a need for a deeper understanding of the chicken crop microbiota since it has an important function in feed digestion and potentially for the health of the chicken. As 1-day-old chickens have a low content of lactobacilli in their crops, it could be beneficial for the chickens to receive lactobacilli early in life. In this study, we analyzed the composition of the crop microbiota able to grow on Lactobacillus-selective (LBS) medium for future selection of candidate probiotic strains. Our results showed that L. reuteri, L. crispatus, and L. salivarius were the most abundant species in the crops of chickens originating from four different farms using two different commercial feeds. The isolates of the most abundant species, L. reuteri, were typed by fatty acid analysis. The distribution of the L. reuteri types varied between the farms. Nisin, a bacteriocin inhibiting mainly gram-positive bacteria (11, 13), may be useful as a cocciodiostat (9). Therefore, nisin resistance of the L. reuteri isolates was also tested in order to evaluate the potential effect of nisin on L. reuteri strains upon feeding chickens nisin-containing feed. Most (76%) of the nisin-sensitive L. reuteri isolates were isolated from the 1-week-old chickens, whereas the majority (87%) of the highly-nisin-resistant strains were obtained from the 5-week-old chickens, suggesting that the L. reuteri population changes as the chickens age.
MATERIALS AND METHODS
Chickens and diets.
One- and 5-week-old broiler chickens with the same genetic background originating from the same hatchery (Broilertalo, Eura, Finland) were obtained from four Finnish farms (designated farm 1 [F1], in Eurajoki, F2, in Halikko, F3, in Lemu, and F4, in Marttila). The killing and sampling of birds were carried out under the Finnish law and statute on animal experiments and followed the ethical principles of the University of Helsinki.
The chickens were fed with two brands (diet A and diet B) of wheat-based diets from two different commercial feed manufacturers, diet A was obtained from Suomen Rehu Ltd. (Espoo, Finland), and diet B was obtained from Raisio Feed Ltd. (Raisio, Finland), with both feeds providing two different wheat-based diets according to the growth phases of the chickens. Starter diets (including, at day 1, Broilact [Orion Corporation, Turku, Finland]) were fed during the first 2 weeks, followed by feeding with the grower diets. Broilact is a competitive-exclusion preparation developed from the cecum content of chickens. Eight different lactobacilli have been isolated from the original inoculum, with one identified as being L. plantarum, one as being L. salivarius, and six as being obligate anaerobes without identification to the species level (Orion Corporation). The main differences between diet A and diet B were that diet A was supplemented with salinomycin (47 mg kg of feed−1) and diet B was supplemented with naracin (50 mg kg of feed−1) coccidiostat during the whole feeding period. Otherwise, the diets were very similar, as the differences in dry matter (6.7 and 5.6% in diet A versus 6.3 and 5.9% in diet B), stoldt fat (7.5 and 7.1% in diet A versus 6.7 and 6.3% in diet B), crude protein (20% in diet A versus 22 and 20% in diet B), and fiber (3 and 3.5% in diet A versus 3.6 and 3.7% in diet B) contents were minor.
Bacterial strains and growth conditions.
L. reuteri CHCC1956 (Christian Hansen Culture Collection, Denmark) served as a type strain. In addition, a human probiotic strain, L. reuteri ATCC 55730 (American Type Culture Collection), was used for the characterization of L. reuteri isolates by fatty acid analysis and fatty acid methyl ester (FAME) chromatography. Lactobacillus isolates were cultivated in deMan, Rogosa, and Sharpe (MRS) medium (Difco Laboratories, Sparks, MD) and in LBS medium (Becton Dickinson Microbiology systems, Cockeysville, MD). The plates were incubated both aerobically and anaerobically (Anaerocult, Merck, Germany) at 41.5°C for 48 h. When required, nisin (Sigma Chemical Co., St. Louis, MO) was used at final concentrations of 10, 20, 30, 50, 100, 200, 300, 400, 500, and 700 IU ml−1.
Isolation and enumeration of crop bacteria.
At 1 and 5 weeks of age, 10 chickens were randomly selected from each flock 2 h after feeding and humanely killed by cervical dislocation. Whole crops were removed from the carcasses and transported on ice to the laboratory. The crop (n = 10) contents were pooled and homogenized in LBS medium (1:10) in a Stomacher laboratory blender (Seward Medical, London, United Kingdom) for 2 min. The crop homogenate was diluted in LBS broth and cultivated by spread plating and pour plating onto LBS agar. The plates were incubated at 41.5°C for 48 h aerobically and anaerobically. For isolation of the crop bacteria located in the vicinity of or associated with the crop epithelium, a 1-cm2 piece from each of the 10 chicken crops (after gravity removal of the digesta and visual inspection to verify a clean mucosa) was cut, placed with the internal sides towards the surface of the LBS agar plates, and incubated overnight at 41.5°C. Before cutting the piece from the crop, the crop was opened with a knife, and the crop digesta was shaken away. The crop digesta dropped easily away from the mucosa, as it was almost solid, revealing the inner epithelium of the crop as being very clean by visual inspection. Bacteria from the obtained lawns of the 10 crop epithelium pieces were resuspended, pooled, and diluted in LBS broth, followed by plating onto LBS agar in order to obtain single colonies. The lactobacilli of the corresponding pooled crop contents were isolated as described above. For partial 16S rRNA gene sequencing, colonies were randomly selected and grown to pure cultures before DNA isolation.
Isolation of DNA.
For the isolation of DNA, bacteria were grown in LBS broth at 37°C for 6 to 8 h and collected by centrifugation. Cells were resuspended in 500 μl of a solution containing 0.01 M EDTA, 0.02 M Tris-HCl (pH 7.5), 0.001 M CaCl2, 0.01 M dithiothreitol, 30 U mutanolysin (Sigma), and 5 mg ml−1 lysozyme. Cells were incubated at 37°C for 1 h, followed by the addition of sodium dodecyl sulfate and proteinase K to final concentrations of 0.5% (wt/vol) and 0.3 mg ml−1. The total volume was increased with water to 700 μl and incubated at 37°C until cell lysis increased the transparency of the solution. The solution was then extracted with phenol-chloroform (1:1) followed by extraction with chloroform-isoamyl alcohol (24:1), ethanol precipitation, washing with 70% ethanol, and resuspension in water after drying (34). The chromosomal DNA thus obtained was used as a template for the PCR amplification of the partial 16S rRNA gene.
Identification of the crop microbiota.
Identification of the isolates was based on partial 16S rRNA gene sequencing. To obtain the sequence of the partial region of the 16S rRNA gene, purified chromosomal DNA (46) from the isolates served as a template in PCR. The amplified area was defined by universal primers pA (5′-AGA GTT TGA TCC TGG CTC AG −3′) and pE′ (5′-CCG TCA ATT CCT TTG AGT TT-3′), which hybridize to the 16S rRNA gene at nucleotides 8 to 28 and 928 to 908 in Escherichia coli (17). The PCR started with heating for 3 min at 94°C, followed by annealing for 1 min at 53°C and extension for 90 s at 72°C and by 30 cycles of PCR (consisting of 45 s at 94°C, 60 s at 53°C, and 90 s at 72°C) and ending the last cycle with storage at 4°C. One strand of the amplified 900-bp fragments was sequenced by the Synthesis and Sequencing Laboratory (Institute of Biotechnology, Helsinki, Finland). Sequence homology of >97% were regarded as belonging to the same species (14). The sequences obtained were compared against the National Center for Biotechnology Information genome BLAST library (version 2.2.8; http://www.ncbi.nlm.nih.gov/BLAST/ ).
Fatty acid analysis and FAME chromatography.
The extraction and derivatization method was done as described previously by Miller (32) and according to the protocol of the Sherlock microbial identification system (MIDI Inc.). Briefly, approximately 40 mg (wet weight) of cells was scraped from the surface of the agar and transferred into a tube with a Teflon-lined cap. Cells were saponified by heating the cells at 100°C/30 min following the addition of 1 ml of 15% NaOH in 50% aqueous methanol. The hydrolysate was cooled, 2 ml of methanolic HCl was added, and the mixture was heated at 80°C for 10 min. The methylated fatty acids were quickly cooled and extracted through the addition of 1.25 ml of hexane-methyl-tert-butyl ether (1:1, vol/vol) with end-end mixing. The phases were allowed to separate, the lower aqueous layer was removed, and 3 ml of dilute NaOH was added to the remaining organic layer. To further clarify the phase interface, saturated NaCl was added. Approximately two-thirds of the organic layer (containing FAMEs) was transferred to a septum-capped vial for analysis. MIDI gas chromatography runs were calibrated against a standard mixture of known fatty acids provided by MIDI. Detected sample peaks were named by interpolation of the retention time using the equivalent chain length method. Peaks that did not display equivalent chains were unnamed. Two microliters of the fatty acid methyl esters was then analyzed using a 5890 A gas chromatograph fitted with a 5% phenyl methyl silicone capillary column, a flame ionization detector, an automatic sampler, and an integrator (Agilent Technologies, Diegem, Belgium) with the Microbial Identification system.
MIC of nisin.
L. reuteri isolates from the chicken crop were grown anaerobically at 37°C using the Bioscreen C system (Labsystem, Finland). From 2 ml of the broth cultured overnight, 1% of the inoculum was made in fresh MRS broth. The bacterial suspension was transferred into the wells (21 wells per inoculum equals three parallel) of bioscreen honeycomb plates. The wells contained 50 μl of 1% inoculum, 200 μl of MRS broth, and 50 μl of nisin suspension with specified concentrations to yield final nisin concentrations of 10, 20, 30, 50, 100, 200, 300, 500, and 700 IU ml−1. The change in turbidity was monitored automatically every 30 min for 72 h at 37°C using a wide-band filter (wide band, absorbance at 420, 450, 492, 540, and 580 nm). The MIC of nisin was regarded as the lowest concentration at which no growth was observed.
Statistical analysis.
RESULTS
Enumeration of the total lactobacilli in the crop contents and in the feed.
The bacterial populations of the crop contents from the broiler chickens growing on LBS plates varied between log 8.08 ± 0.48 and log 8.54 ± 0.30 CFU per gram in the birds 1 and 5 weeks of age, respectively. The results were averaged from numbers obtained from plates incubated aerobically and anaerobically, as the presence of air had insignificant effects on the bacterial counts.
Characterization of the crop microbiota.
About 50 colonies were randomly selected from one plate per pooled crop digesta sample or pooled crop epithelium-associated flora. The visual outlooks of the colonies were very similar in most of the plates, making randomization easy. If the plate contained colonies of different sizes/colors/morphologies, colonies with the different appearance were picked in the same ratio as they existed in the plate.
Approximately 900 bp of the 16S rRNA gene from the randomly selected crop bacteria isolated from LBS was sequenced to identify the isolates to the species level. Nearly 98% of the sequences belonged to the genus Lactobacillus, whereas all the other ones were identified as being Pediococcus acidolactici. The three most abundant Lactobacillus species detected in the crop samples were L. reuteri (33%), L. crispatus (18.7%), and L. salivarius (13.3%). According to the 97% sequence homology criteria, 27.7% of the isolates could not be assigned to any species, but the majority were closely related to L. reuteri, L. crispatus, or L. salivarius. Of these isolates, 34 (seven subtypes) were most related to L. reuteri, 31 (nine subtypes) were most related to L. salivarius, 13 (five subtypes) were most related to L. crispatus, 3 (two subtypes) were most related to L. acidophilus, and 2 (two subtypes) were most closely related to Lactobacillus species oral clone CX36. Some other Lactobacillus spp. were also identified to the species level, but they represented a minority (2.6%) of the isolated Lactobacillus strains (Table 1). The distribution of the L. reuteri, L. crispatus, and L. salivarius strains from the 1- and 5-week-old chickens from the four flocks fed with the two different feeds is shown in Fig. 1. Regardless of the farmhouse, the feed, and the age of the chickens, the L. reuteri, L. crispatus, and L. salivarius species were the most abundant Lactobacillus species. The chickens fed with diet A had significantly more L. salivarius isolates than the chickens fed with diet B (P < 0.05). The higher presence of L. crispatus in the crops of the chickens fed with diet B was almost significant (P = 0.057). L. reuteri was the most abundant species in the crops of the 1-week-old chickens. However, the amount of L. reuteri was reduced significantly in the crops of the 5-week old chickens (P = 0.016). Lactobacillus isolates from the vicinity of the inner epithelium surface of the crops of chickens from F4 were also identified and compared to the Lactobacillus isolates from the corresponding crop contents (Fig. 2) showing similar distributions.
Fatty acid analysis of L. reuteri strains from chicken crop.
As L. reuteri was the most abundant species detected in the crops, we focused on this species. The diversity of L. reuteri isolates was analyzed by gas chromatographic analysis of the whole-cell fatty acids and showed that the 94 isolates appeared to have similar fatty acid compositions. Results disclosed unknown fatty acid peaks and several sum features consisting mainly of different 18:2;18:1;16:0 fatty acid chains and 3-hydroxydecanoic acid as the major 3-OH component in all the isolates. When matching the Euclidian distances (EDs) to one another, tested strains were clearly similar, falling into the same species category (ED < 25). In addition, the fatty acid analysis profiles showed that the isolates could be divided into several clusters (Fig. 3).
Nisin sensitivity of the L. reuteri isolates.
We previously made a food-grade vector based on nisin selection suitable for Lactobacillus spp. (40). L. reuteri isolates are potential targets for genetic engineering. Therefore, we analyzed the nisin sensitivity of the L. reuteri isolates from crops. In addition, nisin-resistant strains could be used to maintain L. reuteri in the crop if chickens have a diet including nisin as a cocciodiostat. Results from the nisin sensitivity analysis (Fig. 3) showed that the nisin sensitivity of the L. reuteri isolates varied from very sensitive (no growth with 50 IU ml−1of nisin) to very resistant (growing at 500 IU ml−1of nisin). The nisin-sensitive isolates were more frequently found (P < 0.05) in younger chickens (77%) than in the 5-week-old chickens (23%). Isolates originating from the chickens fed with diet B had a higher proportion (P < 0.05) of the nisin-resistant isolates (73%) than did chickens fed with diet A (45%).
DISCUSSION
It has been proposed that the crop microbiota acts as a bacterial inoculum for the remainder of the gut (20, 30). Edelman et al. (16) showed that some Lactobacillus isolates colonize the crop and simply pass through the other parts of the chicken intestine. Therefore, knowledge of the composition of the crop microbiota is critical for understanding the contribution of the microbiota members to the well-being of the avian host and for the selection of probiotics (7).
Lactobacilli have an important role in the crop since they are present in large numbers (8). In this study, the numbers of lactobacilli in the chickens' crops were estimated by the cultivation of the crop contents on LBS agar. The amounts of Lactobacillus spp. were in the same range as those obtained (108 to 109 CFU g of crop contents−1) previously by Guan et al. (22), showing that the crop had a high content of Lactobacillus spp.
We identified isolates from the crops of the 1- and 5-week-old chickens to the species level. The isolated crop microbiota was comprised mainly of the three abundant Lactobacillus species (65%): Lactobacillus reuteri, L. crispatus, and L. salivarius. Many Lactobacillus isolates (30%) did not show 16S RNA gene sequence homology high enough (>97%) to be assigned to any defined species, but almost all (92.2%) of them showed the highest homology to either L. reuteri, L. crispatus, or L. salivarius. This result clearly showed that these three Lactobacillus species and some closely related ones dominated the Lactobacillus microbiota of the crop. Some other Lactobacillus spp. were also identified, but they represented the minority of the Lactobacillus isolates. Most of the early studies on the chicken intestinal microbiota, done prior to the reclassification of L. acidophilus, reported that L. reuteri, L. acidophilus, and L. salivarius inhabited the crop and were present throughout the digestive tract (30, 35). As L. acidophilus has been divided into two DNA homology groups containing six related species (18, 24) and L. acidophilus and L. crispatus both belong to the same group, our results are in good concordance with those early studies. Furthermore, it provides some further details by showing that it is the L. crispatus species in the acidophilus group that is characteristic of this habitat. When the composition of the crop microbiota of this study is compared to that reported in a more recent study (22), having more precise species determination, some differences can be seen. Using denaturing gradient gel electrophoresis and amplified rRNA gene restriction analysis, Guan et al. (22) showed that the crop microbiota varied in composition during the lives of the chickens, with some species, such as L. acidophilus and L. salivarius, appearing in a developmental succession, while other species, i.e., L. reuteri, L. johnsonii, and one or more of the species L. crispatus, L. gallinarum, and L. amylovorus, were consistently detected. L. cripatus and L. acidophilus were the most dominant species at 1 week of age, and L. salivarius was the most dominant at 5 weeks of age (22). In our study, L. reuteri was clearly dominating only in 1-week-old chickens (P < 0.005), whereas the dominance of L. salivarius in 5-week-old chickens was related to the feed (P < 0.05). The competitive-exclusion preparation, which includes L. salivarius but not L. reuteri and L. crispatus and which was given to the chickens on the first day, may be a source of L. salivarius for the chickens but does not determine the abundance of this species in the crop. L. reuteri, L. crispatus, and L. salivarius appear to be dominating lactobacilli in the chicken crops, but which species is the most dominant at the moment of analysis may depend on the age and the diet of the chickens. In contrast to the study described previously by Guan et al. (22), L. acidophilus was not a dominating species in the chicken crop at all. This may be due to differences caused by the different origins and locations of the chickens or a diet effect. Another explanation is that it was an effect of the competitive-exclusion preparation that the chickens in our study received but that those in the study described previously by Guan et al. (22) did not.
Isolates originating from the inner surfaces of the crops were also identified and compared to the crop contents, showing that the species abundances and distributions were very similar at both locations. This suggests that the Lactobacillus microbiota of the crop content may arise from the inoculum of the bacteria originating from the inner crop surface. These bacteria potentially adhere to the epithelium and shed to the crop content. This could also be a possible explanation for why the lactobacillus composition is so similar in the crops of the chickens analyzed in different countries; in other words, the epithelium of the crop and the conditions inside the crop may be more important factors defining the crop microbiota than the bacterial intake via feed or the feed composition. The result that the different feeds in our study did not give rise to any new dominating Lactobacillus species outside of the typical L. reuteri, L. cripatus, L. salivarius, and L. acidophilus chicken crop species further suggests a minor role of the feed as an introducer of new dominating bacteria into the crop.
Our results show that the most abundant Lactobacillus species in the 1-week-old chicken crops was L. reuteri. Therefore, we focused on this species by characterizing the diversity of the isolates using fatty acid and nisin resistance analyses. The fatty acid analysis showed that the crop L. reuteri isolates could be divided into several clusters, suggesting that several different strains of L. reuteri may be present in the crop. Previously, no studies on the strain diversity of any chicken crop species were made. Our results clearly show that for a deeper understanding of the crop microbiota, information on strain diversity may be important, as the distribution of the L. reuteri isolates into clusters varied between the chickens originating from the different farms.
The results from the nisin resistance analysis of the L. reuteri isolates further strengthened the view that the L. reuteri strains of the chickens from the different farms vary. Feeding with diet B favored nisin-resistant L. reuteri strains, as the proportion of nisin-resistant strains was significantly higher in the crops of these chickens than in crops of chickens fed with diet A. One difference between the diets was in the coccidiostats used. As salinomycin in diet B is known to negatively influence lactic acid bacteria (18, 24), it is possible that salinomycin selected for L. reuteri strains with mutations resulting in nisin resistance as a pleiotropic effect. As both antimicrobials are membrane-active agents, cross-effects of resistance mechanisms may be possible (15, 21). The result that more nisin-resistant isolates were found in the older chickens than in 1-week-old chickens showed that the L. reuteri population in the chickens changes during aging. This change in the nisin resistance of L. reuteri isolates suggests that adding nisin to feed as a potential growth promoter may negatively affect the L. reuteri population of young chickens, whereas older chickens may be less influenced. On the other hand, the nisin-resistant L. reuteri strains isolated in this study could be added to nisin-containing feed fed to young chickens, thereby potentially maintaining L. reuteri as part of the dominating crop microbiota. The nisin-sensitive L. reuteri isolates could be used as hosts for the transformation of plasmids using food-grade nisin selection. However, the colonization of such engineered strains in chickens using nisin selection pressure could be difficult in the older chickens.
The genus Lactobacillus is essential for modern food and feed technologies. They are consumed by humans and fed to animals of commercial value as probiotics in efforts to maintain a balanced microbiota and to reduce the numbers of potential pathogens residing in the intestinal tract (26, 33, 44). L. reuteri and several others intestinal Lactobacillus strains have been widely used as probiotics (10, 26, 30). Our results showing the dominance of L. reuteri in the crop further suggest that it may be a good choice to include L. reuteri strains in future studies to find probiotic effects of bacterial supplements given to chickens.
FIG. 1.
FIG. 2.
FIG. 3.
TABLE 1.
| Speciesa | No. of isolates | % of isolates | No. of times isolates were found in 8 crop samples |
|---|---|---|---|
| L. acidophilus/L. johnsonii | 5 | 1.7 | 4 |
| L. crispatus | 56 | 18.7 | 7 |
| L. gallinarum | 1 | 0.3 | 1 |
| L. helveticus | 1 | 0.3 | 1 |
| L. pentosus | 1 | 0.3 | 1 |
| L. reuteri | 99 | 33.0 | 7 |
| L. salivarius | 40 | 13.3 | 7 |
| Lactobacillus sp. oral clone CX36 | 6 | 2.0 | 3 |
| Lactobacillus sp. strain CLE-4 | 1 | 0.3 | 1 |
| Lactobacillus spp.b | 83 | 27.7 | 8 |
| P. acidolactici | 7 | 2.3 | 2 |
| Total | 300 | 100 | 8 |
a
Identification of species was based on partial 16S RNA gene sequencing and comparison of sequences obtained against the National Center for Biotechnology Information genome BLAST library. Homology of >97% was used as the criterion for species identification. Isolates from the vicinity of the crop epithelium are also included.
b
Lactobacillus spp. which could not be assigned to any species, as their sequence homologies to known species were <97%.
Acknowledgments
We are deeply indebted to Irina Tisko, who assisted in the fatty acid analysis, and to Seppo Peuranen for his help in sourcing the chickens for the study.
This study was supported by Danisco Cultor Innovation Ltd., Kantvik, Finland, and the Academy of Finland, project number 177321.
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Published In
Applied and Environmental Microbiology
Volume 73 • Number 24 • 15 December 2007
Pages: 7867 - 7873
PubMed: 17933935
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© 2007.
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Received: 21 May 2007
Accepted: 30 September 2007
Published online: 15 December 2007
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2007. Identification of the Most Abundant Lactobacillus Species in the Crop of 1- and 5-Week-Old Broiler Chickens. Appl Environ Microbiol 73:.
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