Quantitative detection of Clostridium perfringens in the broiler fowl gastrointestinal tract by real-time PCR

Strains of Clostridium perfringens are a frequent cause of food-borne disease and gas gangrene and are also associated with necrotic enteritis in chickens. To detect and quantify the levels of C. perfringens in the chicken gastrointestinal tract, a quantitative real-time PCR assay utilizing a fluorogenic, hydrolysis-type probe was developed and utilized to assay material retrieved from the broiler chicken cecum and ileum. Primers and probe were selected following an alignment of 16S rDNA sequences from members of cluster I of the genus Clostridium, and proved to be specific for C. perfringens. The assay could detect approximately 50 fg of C. perfringens genomic DNA and approximately 20 cells in pure culture. Measurements of the analytical sensitivity determined with spiked intestinal contents indicated that the consistent limit of detection with ileal samples was approximately 10(2) CFU/g of ileal material, but only about 10(4) CFU/g of cecal samples. The decreased sensitivity with the cecal samples was due to the presence of an unidentified chemical PCR inhibitor(s) in the cecal DNA purifications. The assay was utilized to rapidly detect and quantify C. perfringens levels in the gut tract of broiler chickens reared without supplementary growth-promoting antibiotics that manifested symptoms of necrotic enteritis. The results illustrated that quantitative real-time PCR correlates well with quantification via standard plate counts in samples taken from the ileal region of the gastrointestinal tract.     

Multiplex PCR assay for toxinotyping Clostridium perfringens isolates obtained from Finnish broiler chickens

AIMS: The aim of the study was to determine the presence of genes coding for alpha (cpa), beta (cpb), epsilon (etx), iota (iA) and enterotoxin (cpe) from Clostridium perfringens broiler chicken isolates, using multiplex PCR assay established in the study. METHODS AND RESULTS: The multiplex PCR assay was shown to be specific when tested with 10 C. perfringens strains representing different toxin types, and 15 strains of other bacterial species. All 118 broiler chicken C. perfringens isolates were shown to carry the cpa gene but not cpb, etx, iap or cpe genes, signifying that all isolates represented type A and were cpe-negative. CONCLUSIONS: The assay established in the study enables the simultaneous detection of the major toxin genes and the cpe gene from C. perfringens isolates. SIGNIFICANCE AND IMPACT OF THE STUDY: The present study offers a new primer pair for detecting cpa, combined with a multiplex PCR assay. In addition, the study provides data of the presence of different toxin genes in C. perfringens isolates obtained from broiler chickens.     

Development of real-time PCR for the detection of Clostridium perfringens in meats and vegetables

A real-time PCR assay was developed and validated inhouse specifically for the detection of Clostridium perfringens (Cl. perfringens) in meats and vegetables by comparing with the culture method. The detection limit of the real-time PCR assay in phosphate-buffered saline was 102 CFU/ml. When the two methods were compared in food samples inoculated with Cl. perfringens, the culture method detected 52 positives, whereas real-time PCR detected 51 positives out of 160 samples. The difference was without statistical significance (p>0.05). Real-time PCR assay is an option for quality assurance laboratories to perform standard diagnostic tests, considering its detection ability and time-saving efficiency.     

Enumeration of specific bacterial populations in complex intestinal communities using quantitative PCR based on the chaperonin-60 target

We used qPCR and the target gene chaperonin-60 (cpn60) to enumerate Clostridium perfringens genomes in DNA extracts from contents of the chicken gastrointestinal tract with the aim of optimizing this methodology to enumerate any bacterium of interest. To determine the most accurate protocols for determining target species abundance, we compared various DNA extraction methods in combination with four methods for producing standard curves. Factors affecting accuracy included the co-purification of PCR inhibitors and/or fluorescence quenchers and the yield of target DNA in the extract. Anion exchange chromatography of the spiked test samples enabled accurate enumeration of C. perfringens using a standard curve comprised of a plasmid containing a fragment of C. perfringens cpn60. We used qPCR to enumerate C. perfringens and other intestinal bacteria in ileum and cecum samples from chickens that had been challenged with C. perfringens and compared the results with viable counts on corresponding selective agars. We conclude that qPCR-based molecular enumeration of target species in the gastrointestinal tract is feasible, but care must be taken in order to mitigate the effects of confounding factors that can affect the apparent cell count.     

Comparison of agar plate and real-time PCR on enumeration of Lactobacillus, Clostridium perfringens and total anaerobic bacteria in dog faeces

AIMS: To compare agar plate and real-time PCR methods on enumeration of total anaerobic bacteria, Lactobacillus and Clostridium perfringens in dog faeces. METHODS AND RESULTS: Thirty-two faecal specimens from Labrador retriever dogs were used to compare agar plate and real-time PCR enumeration methods for Lactobacillus, C. perfringens and total anaerobic bacteria. Total anaerobic bacteria, C. perfringens and Lactobacillus of faeces were counted (as CFU g(-1) faeces) for 48-h incubation at 37 degrees C in an anaerobic gas chamber on genus-selective media. Total genomic DNA from samples was extracted by the QIAamp DNA stool mini kit. The quantification of DNA (as DNA copy per gram faeces) by real-time PCR was performed with a LightCycler system with the QuantiTect SYBR green PCR kit for PCR amplification. The results indicated that there was a significant correlation between CFU and DNA copy of Lactobacillus (R2 = 0.78, P < 0.01) and total anaerobic bacteria (R2 = 0.21, P < 0.05); but no correlation was found between CFU and DNA copy of C. perfringens. The regression equations for Lactobacillus and total anaerobic bacteria were log(DNA copy) = 0.83 x log(CFU) + 1.43 and log(DNA copy) = 1.62 x log(CFU) - 6.32 respectively. CONCLUSIONS: The real-time PCR method could be used to enumerate Lactobacillus within 2 days when compared with plating method which requires 5-6 days. SIGNIFICANCE AND IMPACT OF THE STUDY: The real-time PCR method and the primer set for Lactobacillus spp. harboured in the dog intestine can be used for rapid enumeration of lactobacilli and monitoring of the faecal Lactobacillus community.     

A real-time PCR for the detection of Salmonella Enteritidis in poultry meat and consumption eggs

A robust duplex 5' nuclease (TaqMan) real-time PCR was developed and in-house validated for the specific detection of Salmonella enterica subspecies enterica serovar Enteritidis in whole chicken carcass rinses and consumption eggs. The assay uses specifically designed primers and a TaqMan probe to target the Prot6e gene located on the S. Enteritidis specific 60-kb virulence plasmid. As an internal amplification control to monitor Salmonella DNA in the sample, a second primer/TaqMan probe set detects simultaneously the Salmonella specific invA gene. The assay identified correctly 95% of the 79 Salmonella Enteritidis strains tested comprising 19 different phage types. None of the 119 non-Enteritidis strains comprising 54 serovars was positive for the Prot6e gene. The assay detection probability was for 10(2) or more genome equivalents 100% and for 10 equivalents 83%. A pre-PCR sample preparation protocol including a pre-enrichment step in buffered peptone water, followed by DNA extraction was applied on low levels of artificially contaminated whole chicken carcass rinses and eggs from hens as well as 25 potentially naturally contaminated chickens. The detection limit was less than three CFU per 50 ml carcass rinse or 10 ml egg. The sensitivity and specificity compared to the traditional culture-based detection method and serotyping were both 100%. Twenty-five potentially naturally contaminated chickens were compared by the real-time PCR and the traditional cultural isolation method resulting in four Salmonella positive samples of which two were positive for the Prot6e gene and serotyped as S. Enteritidis. We show also that Salmonella isolates which have a rough lipopolysaccharide structure could be assigned to the serovar Enteritidis by the real-time PCR. This methodology can contribute to meet the need of fast identification and detection methods for use in monitoring and control measures programmes.     

Effects of dietary fat source and subtherapeutic levels of antibiotic on the bacterial community in the ileum of broiler chickens at various ages

The effect of dietary fat source (soy oil or a mixture of lard and tallow) and dietary supplementation with antibiotics (a combination of avilamycin at 10 mg kg of feed(-1) and salinomycin at 40 mg kg of feed(-1)) on the bacterial community in the ileum of broiler chickens at different ages (7, 14, 21, and 35 days) was studied using PCR with denaturing gradient gel electrophoresis (DGGE) analysis and bacteriological culture. The bacterial origin of fragments in DGGE profiles was identified by sequencing. Bacterial enumeration results, together with PCR-DGGE profiles, showed that the composition of the microflora was age dependent and influenced by dietary fat source and antibiotic supplementation. An increased incidence of streptococci, enterobacteria, and Clostridium perfringens with age of the chickens was demonstrated. Lactobacilli and C. perfringens were the bacterial groups most strongly affected by the dietary treatments. Moreover, different strains (clonal variants of the alpha-toxin gene) of C. perfringens type A were detected in response to age, dietary fat source, and dietary supplementation with antibiotics.     

APPLICATION OF A COLONY PCR TECHNIQUE WITH FUNG'S DOUBLE TUBE METHOD FOR RAPID DETECTION AND CONFIRMATION OF CLOSTRIDIUM PERFRINGENS

A colony polymerase chain reaction (PCR) technique was applied with an established Fung's double tube (FDT) method for rapid detection and confirmation of Clostridium perfringens. Published sequences of PCR primers for C. perfringens alpha toxin gene were used and PCR conditions were optimized. From the detection of C. perfringens by FDT tube to the confirmation by a colony PCR assay took as short as 16–18 h. The method was applied to 147 isolates of anaerobic sulfite reducing bacteria isolated from foods, sewages and animal clinical specimens. The results were compared with standard methods for the confirmation of C. perfringens. Of those 147 suspected isolates, 97 and 99 were confirmed as C. perfringens by standard methods and the colony PCR technique, respectively. We found the developed method simple, rapid, cost-effective, and most importantly, very reliable for the detection and confirmation of C. perfringens.

PRACTICAL APPLICATIONS

With Fung's double tube method and a colony polymerase chain reaction technique, a completed determination of Clostridium perfringens contamination can be accomplished within 16–18 h. This saves at least 2–3 days when compared with standard methods. Moreover, it minimizes the cost and labor needed since an anaerobic chamber as well as steps of Gram staining and biochemical testing can be avoided. The developed method is a powerful tool and an alternative for the enumeration of C. perfringens . It can be applied to samples from various sources and highly reliable results are expected. Most microbiological laboratories have a thermocycler and reagents as parts of their basic instruments. Therefore, the developed method can be easily applied without massive investment.     

Quantitative Detection of Clostridium perfringens in the Broiler Fowl Gastrointestinal Tract by Real-Time PCR

Strains of Clostridium perfringens are a frequent cause of food-borne disease and gas gangrene and are also associated with necrotic enteritis in chickens. To detect and quantify the levels of C. perfringens in the chicken gastrointestinal tract, a quantitative real-time PCR assay utilizing a fluorogenic, hydrolysis-type probe was developed and utilized to assay material retrieved from the broiler chicken cecum and ileum. Primers and probe were selected following an alignment of 16S rDNA sequences from members of cluster I of the genus Clostridium, and proved to be specific for C. perfringens. The assay could detect approximately 50 fg of C. perfringens genomic DNA and approximately 20 cells in pure culture. Measurements of the analytical sensitivity determined with spiked intestinal contents indicated that the consistent limit of detection with ileal samples was approximately 10² CFU/g of ileal material, but only about 10⁴ CFU/g of cecal samples. The decreased sensitivity with the cecal samples was due to the presence of an unidentified chemical PCR inhibitor(s) in the cecal DNA purifications. The assay was utilized to rapidly detect and quantify C. perfringens levels in the gut tract of broiler chickens reared without supplementary growth-promoting antibiotics that manifested symptoms of necrotic enteritis. The results illustrated that quantitative real-time PCR correlates well with quantification via standard plate counts in samples taken from the ileal region of the gastrointestinal tract.     

产气荚膜梭菌实时荧光PCR方法的建立

目的:利用荧光定量PCR技术,建立快速敏感特异的检测产气荚膜梭菌的方法。方法:以产气荚膜梭菌基因为靶序列设计引物和探针,以自产气荚膜梭菌菌株中提取的DNA为模板,优化引物和探针的浓度比,同时验证方法的特异性、敏感性。结果:建立的反应体系在上游引物浓度为0.45μmol/L、下游引物浓度为0.15μmol/L、探针浓度为0.3μmol/L时,具有良好的特异性和敏感性,与创伤弧菌等12种相关细菌均无交叉反应;对纯菌检测的灵敏度低于10 CFU/反应体系。结论:建立的实时荧光PCR方法特异、灵敏、快速,能对战时气性坏疽做出快速准确的报告,实现对这种战时高发疾病的安全、快速和定量检测。     

Necrotic enteritis challenge models with broiler chickens raised on litter: evaluation of preconditions, Clostridium perfringens strains and outcome variables

The effect of Clostridium perfringens challenge, number of challenge days, and pre-challenge antibiotic treatment on the induction of necrotic enteritis in broiler chickens raised on litter was studied, and the relationship between bacterial counts and frequency of gut lesions was evaluated. Specific intestinal lesions in randomly selected birds were present despite a lack of disease-specific mortality. Challenge, number of challenge days and frequency of lesions were associated with median counts of C. perfringens. The effect of pre-challenge C. perfringens counts and antibiotics cannot be evaluated unless procedures for the control of pre-challenge infection and methods for the differentiation between wild-type and challenge strains are established.     

Real-time quantitative PCR measurement of ileal Lactobacillus salivarius populations from broiler chickens to determine the influence of farming practices

A real-time quantitative PCR assay targeting a 16S-23S intergenic spacer region sequence was devised to measure the sizes of populations of Lactobacillus salivarius present in ileal digesta collected from broiler chickens. This species has been associated with deconjugation of bile salts in the small bowel and reduced broiler productivity. The assay was tested as a means of monitoring the sizes of L. salivarius populations from broilers fed diets with different compositions, maintained at different stocking densities, or given the antimicrobial drugs bacitracin and monensin in the feed. Stocking densities did not influence the numbers of L. salivarius cells in the ileum. A diet containing meat and bone meal reduced the size of the L. salivarius population relative to that of chickens given the control diet, as did administration of bacitracin and monensin in the feed. These changes in the target bacterial population were associated with improved broiler weight gain.     

Quantitative analysis of the intestinal bacterial community in one- to three-week-old commercially reared broiler chickens fed conventional or antibiotic-free vegetable-based diets

AIMS: To explore the effect of drug-free poultry production on the intestinal microflora of broiler chickens, the bacterial community of this environment was quantitatively profiled in both conventionally reared birds and birds reared without antibiotic growth promotants (AGPs) on a vegetable-based diet. METHODS AND RESULTS: Quantitative, real-time PCR with group-specific 16S rDNA primer sets was used to enumerate the abundance of the following chicken gastrointestinal (GI) tract phylogenetic groups: the Clostridium leptum-Faecalibacterium prausnitzii subgroup (Clostridium genus cluster IV), the Clostridium coccoides - Eubacterium rectale subgroup (Clostridium cluster XIVa and XIVb), the Bacteroides group (including Prevotella and Porphyromonas), Bifidobacterium spp., the Enterobacteriaceae, the Lactobacillus group (including the genera Leuconostoc, Pediococcus, Aerococcus and Weissella), the Clostridium perfringens subgroup (Clostridium cluster I), Enterococcus spp., Veillonella spp., Atopobium spp., Campylobacter spp. and the domain Bacteria. A species-specific 5'-nuclease (Taqman) assay was also employed to specifically assess Cl. perfringens abundance. Ten birds were sampled from each of two commercial chicken houses, one in which feed was supplemented with AGPs and exogenous animal protein, and the other vegetable-based and drug-free, at 7, 14 and 21 days of age. The ileal community was dominated by two large populations, the lactobacilli and the Enterobacteriaceae, with those taxa much more numerous in drug-free vegetable-based diet fed birds than those conventionally reared at the 7- and 14-day time periods. The progressive changes in microflora in both the conventional and drug-free caeca were similar to each other, with the Enterobacteriaceae sequences dominating at day 7, but being replaced by obligate anaerobe signature sequences by day 14. Of note was the finding that all the day 14 and day 21 replicate caecal samples from the drug-free house were positive for Campylobacter spp. averaging >10(8) 16S rDNA gene copies per gram wet weight. CONCLUSIONS: Quantitative, real-time PCR indicates that the effects of drug-free rearing on the chicken GI tract microbial community are most pronounced in the ileal region, but AGPs may be important in controlling Campylobacter colonization of the caecum. SIGNIFICANCE AND IMPACT OF THE STUDY: A quantitative taxonomic understanding of the shifting microbial ecology of the broiler chicken gut microbiota is important in the light of AGP withdrawal. AGP withdrawal has occurred in response to concerns over the transfer of antimicrobial-resistant bacteria to humans via the food production chain.     

Prevalence and characterization of Clostridium perfringens from spices in Argentina

Spices can present high microbial counts and Clostridium perfringens, Bacillus cereus, Salmonella and Shigella, among others have been isolated from spices. C. perfringens is an important pathogen agent causing, among other diseases, enteritis in humans caused by C. perfringens enterotoxin (CPE) which causes human food poisoning and enterotoxemia in domestic animals. The aims of the present work were (i) to establish the hygienic sanitary quality of some spices in San Luis, Argentina; (ii) to determine the presence of C. perfringens in these spices by means of the most probable number (MPN) and count on plate methods; (iii) to characterize the enterotoxigenic strains of C. perfringens by PCR and immunological methods such as reverse passive latex agglutination (RPLA) and (iv) to type by PCR C. perfringens strains isolated. A total of 115 samples of spices, 67 of which were purchased in local retail stores and 48 domestically collected were analysed. Total aerobe counts on tryptone glucose yeast extract agar medium of the 115 samples were between <10 and 10(6) CFU/g. The colifecal counts using Mac Conkey broth of the 115 samples were <4-10(3)CFU/g, with 28 samples (24.34%) exceeding the limit established by the Spanish Alimentary Code (10 CFU/g) while 2 samples (1.73%) had a sulfite reducing anaerobe load above standard limits. A total of 14 C. perfringens strains (12.17%) were isolated and characterized from 115 samples by the standard biochemical tests. Four of which (28.60%) turned out to be enterotoxigenic by PCR and RPLA. In order to type C. perfringens strains based on their main toxins, the 14 strains were analysed by PCR. All strains belonged to type A. All RPLA positive strains were cpe(+) by PCR. The percentage of enterotoxigenic strains was more elevated that those reported in other studies for this type of sample. These results indicate that sanitary conditions in different production stages of species must be improved to reduce health hazards. The high percentage (24.34%) of samples with colifecal values above standard limits is an indication of deficient sanitary conditions. These results suggest the need to provide legislation on the sanitary and hygienic quality of spices in our country.     

First detection of the microsporidium Enterocytozoon bieneusi in non-mammalian hosts (chickens)

Faecal samples taken from eight underweight, approximately 5-week-old broiler chickens in a poultry abattoir were investigated for microsporidial infections by light microscopy, electron microscopy, and PCR. In two of six chickens, which were suspected of being infected with microsporidia by light microscopy, Enterocytozoon bieneusi (genotype 'J') was detected by PCR and DNA sequencing, and in one of the two PCR-positive samples by extensive electron microscopy. This is the first time that E. bieneusi has been detected in chickens, i.e. in a non-mammalian species.     

Improved real-time PCR estimation of gene copy number in soil extracts using an artificial reference

Application of polymerase chain reaction (PCR) techniques has developed significantly from a qualitative technology to include powerful quantitative technologies, including real-time PCR, which are regularly used for detection and quantification of nucleic acids in many settings, including community analysis where culture-based techniques are not suitable. Many applications of real-time PCR involve absolute quantification which is susceptible to inaccuracies caused by losses during DNA extraction or inhibition caused by co-extracted compounds. We present here an improvement to this approach involving the addition of an artificial internal standard, prior to nucleic acid extraction. The standard was generated by in-situ mutagenesis from an E. coli template to ensure it both did not amplify with bacterial primers used for quantification and was short enough to minimise possible interference with other analyses. By estimating gene target copies by relative abundance, this approach accounts for both loss during extraction and inhibition effects. We present a novel application of relative real time PCR, using the internal standard as a reference, allowing accurate estimation of total bacterial populations both within and across a wide range of soils and demonstrate its improvement over absolute quantification by comparison of both approaches to ester linked fatty acid analysis of the same soils.     

Comparison Between Effects of Standard Feed and Whole Wheat Supplemented Diet on Experimental Eimeria tenella and Eimeria maxima Infections in Broiler Chickens

The effects of experimental infections with Eimeria tenella (Experiment 1, n = 144) or E. maxima (Experiment 2, n = 216) in broiler chickens fed whole wheat, with or without access to grit, as compared to a standard pelleted feed were studied. Inclusion of whole wheat was gradually increased up to 30% at 3 weeks of age. Grit was given separately. The chickens were kept on litter in a parasite-free environment with free access to water and feed. At 3 weeks of age half the number of chickens were individually inoculated with 500 sporulated oocysts of E. tenella (Experiment 1) or 3 000 sporulated oocysts of Eimeria maxima (Experiment 2), and the remaining birds were kept separate as uninfected controls. Neither coccidiostats nor growth enhancers were used. Oocyst concentration was determined from each group separately. Intestinal lesions were scored on 6 birds per feed regime 7 d postinoculation, and on the remaining birds at slaughter. Diet had no significant effect on bird performance during infection. However, there was an indication that the E. maxima infection had more negative effect on weight gain in birds given standard feed than in those given whole wheat supplement, but the difference was not significant (p<0.09). The number of oocysts shed or mean intestinal lesion scores did not differ between diets in either experiment. In both experiments, the number of Clostridium perfringens was higher in the caeca of inoculated birds, but there were no differences between diets.     

New real-time quantitative PCR procedure for quantification of bifidobacteria in human fecal samples

The application of a real-time quantitative PCR method (5' nuclease assay), based on the use of a probe labeled at its 5' end with a stable, fluorescent lanthanide chelate, for the quantification of human fecal bifidobacteria was evaluated. The specificities of the primers and the primer-probe combination were evaluated by conventional PCR and real-time PCR, respectively. The results obtained by real-time PCR were compared with those obtained by fluorescent in situ hybridization, the current gold standard for intestinal microbiota quantification. In general, a good correlation between the two methods was observed. In order to determine the detection limit and the accuracy of the real-time PCR procedure, germfree rat feces were spiked with known amounts of bifidobacteria and analyzed by both methods. The detection limit of the method used in this study was found to be about 5 x 10(4) cells per g of feces. Both methods, real-time PCR and fluorescent in situ hybridization, led to an accurate quantification of the spiked samples with high levels of bifidobacteria, but real-time PCR was more accurate for samples with low levels. We conclude that the real-time PCR procedure described here is a specific, accurate, rapid, and easy method for the quantification of bifidobacteria in feces.     

Genotypic variability at the major histocompatibility complex (B and Rfp-Y) in Camperos broiler chickens

Evidence for the importance of major histocompatibility complex (MHC) genotype in immunological fitness of chickens continues to accumulate. The MHC B haplotypes contribute resistance to Marek's and other diseases of economic importance. The Rfp-Y, a second cluster of MHC genes in the chicken, may also contribute to disease resistance. Nevertheless, the MHC B and Rfp-Y haplotypes segregating in broiler chickens are poorly documented. The Camperos, free-range broiler chickens developed in Argentina, provide an opportunity to evaluate MHC diversity in a genetically diverse broiler stock. Camperos are derived by cross-breeding parental stocks maintained essentially without selection since their founding. We analysed 51 DNA samples from the Camperos and their parental lines for MHC B and Rfp-Y variability by restriction fragment pattern (rfp) and SSCP typing methods for B-G, B-F (class Ia), B-Lbeta (class II) and Y-F (class Ib) diversity. We found evidence for 38 B-G genotypes. The Camperos B-G patterns were not shared with White Leghorn controls, nor were any of a limited number of Camperos B-G gene sequences identical to published B-G sequences. The SSCP assays provided evidence for the presence of at least 28 B-F and 29 B-Lbeta genotypes. When considered together B-F, B-L, and B-G patterns provide evidence for 40 Camperos B genotypes. We found even greater Rfp-Y diversity. The Rfp-Y class I-specific probe, 163/164f, revealed 44 different rfps among the 51 samples. We conclude that substantial MHC B and Rfp-Y diversity exists within broiler chickens that might be drawn upon in selecting for desirable immunological traits.     

Real-Time Quantitative PCR Measurement of Ileal Lactobacillus salivarius Populations from Broiler Chickens To Determine the Influence of Farming Practices

A real-time quantitative PCR assay targeting a 16S-23S intergenic spacer region sequence was devised to measure the sizes of populations of Lactobacillus salivarius present in ileal digesta collected from broiler chickens. This species has been associated with deconjugation of bile salts in the small bowel and reduced broiler productivity. The assay was tested as a means of monitoring the sizes of L. salivarius populations from broilers fed diets with different compositions, maintained at different stocking densities, or given the antimicrobial drugs bacitracin and monensin in the feed. Stocking densities did not influence the numbers of L. salivarius cells in the ileum. A diet containing meat and bone meal reduced the size of the L. salivarius population relative to that of chickens given the control diet, as did administration of bacitracin and monensin in the feed. These changes in the target bacterial population were associated with improved broiler weight gain.