Combination of probiotics and coccidiosis vaccine enhances protection against an challenge

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Development of a Strain-Specific Real-Time PCR Assay for Enumeration of a Probiotic <i>Lactobacillus reuteri</i> in Chicken Feed and Intestine

<div><p>A strain-specific real-time PCR assay was developed for quantification of a probiotic <i>Lactobacillus reuteri</i> (DSM 16350) in poultry feed and intestine. The specific primers were designed based on a genomic sequence of the strain derived from suppression subtractive hybridization with the type strain <i>L. reuteri</i> DSM 20016. Specificity was tested using a set of non-target strains from several sources. Applicability of the real-time PCR assay was evaluated in a controlled broiler feeding trial by using standard curves specific for feed and intestinal matrices. The amount of the probiotic <i>L. reuteri</i> was determined in feed from three feeding phases and in intestinal samples of the jejunum, ileum, and caecum of three, 14, and 39 day old birds. <i>L. reuteri</i> DSM 16350 cells were enumerated in all feeds supplemented with the probiotic close to the inclusion rate of 7.0×10³ cfu/g, however, were not detected in <i>L. reuteri</i> DSM 16350 free feed. In three day old birds <i>L. reuteri</i> DSM 16350 was only detected in intestinal samples from probiotic fed animals ranging from 8.2±7.8×10⁵ cfu/g in the jejunum, 1.0±1.1×10⁷ cfu/g in the ileum, and 2.5±5.7×10⁵ cfu/g in the caecum. Similar results were obtained for intestinal samples of older birds (14 and 39 days). With increasing age of the animals the amount of <i>L. reuteri</i> signals in the control animals, however, also increased, indicating the appearance of highly similar bacterial genomes in the gut microbiota. The <i>L. reuteri</i> DSM 16350 qPCR assay could be used in future for feeding trials to assure the accurate inclusion of the supplement to the feed and to monitor it's uptake into the GIT of young chicken.</p></div

Mean numbers of <i>L. reuteri</i> LR in chicken gut samples from animals fed without (control) or with LR (probiotic) over 39 days as determined by strain-specific qPCR.

<p>n.d. not detected above detection limit for gut sample 4×10² cfu/g.</p><p>n number of animals with cell counts above detection limit from a total number of eight animals.</p

qPCR standard curve for quantification of <i>L. reuteri</i> (LR) in feed and gut samples.

<p>The standard curves were generated by amplification of serially diluted DNA from feed spiked with 8.0×10⁸ cfu/g and from gut lumen content spiked with 8.5×10⁶ cfu/g.</p