The Preliminary Development of an in vitro Poultry Cecal Culture Model to Evaluate the Effects of Original XPCTM for the Reduction of Campylobacter jejuni and Its Potential Effects on the Microbiota

Abstract

Poultry is a major reservoir for the pathogen Campylobacter jejuni. C. jejuni inhabits the poultry gastrointestinal tract as a part of the gut microbiota. The objective of this study was to evaluate both the survival of C. jejuni and the changes in the population dynamics of the cecal microbiome during an in vitro C. jejuni inoculation in the presence or absence of the functional metabolites of Diamond V Original XPC TM (XPC). Two independent trials were conducted. Broiler chickens (n = 6 per Trial 1 and n = 3 per Trial 2) were raised according to standard industry guidelines and euthanized on Day 41. The ceca were collected aseptically, their contents removed independently and then used in an in vitro microaerobic model with 0.1% cecal contents + Campylobacter with or without 1% XPC (w/v). Before the inoculation with a chloramphenicol resistant marker strain of C. jejuni, the cecal contents were pre-incubated with XPC at 42 • C for 24 h, in a shaking incubator (200 rpm) under microaerobic conditions, then experimentally inoculated with 10 8 /ml of C. jejuni into the appropriate treatment groups. At 0 and 24 h for Trial 1, and 48 h for Trial 2, sub-samples of the culture (n = 3 ceca, two technical replicates per ceca, XPC alone or ceca culture alone) were enumerated using a Petroff-Hausser counter, and the DNA was extracted for microbiome analysis. DNA was isolated using the Qiagen QIAamp Fast Stool DNA Mini Kit and sequenced using the Illumina MiSeq platform. The reads were filtered, normalized, and assigned taxonomical identities using the QIIME2 pipeline. The relative microbiota populations were identified via ANCOM. Altogether, evidence suggests that XPC alters the microbiome, and in turn reduces Campylobacter survival.

The biological effects of microencapsulated organic acids and botanicals induces tissue-specific and dose-dependent changes to the microbiota

“…One specific limitation to the current study is that the microbiome results are qualitative which did not allow us to directly consider cell counts and 16S rDNA copy number. Future studies should focus on using a greater statistical power for compositional analyses [39,40]. Different populations can contribute varying copy numbers of 16S rDNA to the analysis; therefore, using a quantitative method may be more important in the future [41].…”

“…The DNA was extracted and sequenced as per standard laboratory guidelines [40]. Briefly, the tissue (ileum or jejunum) contents were thawed, homogenized, and 0.3 g removed followed by extraction using the Qiagen Stool Kit (Qiagen, Hilden, Germany).…”

“…Using the amplicon sequence variance index primers and protocol, the library was prepared as previously described [44]. Normalization and library clean-up were also performed prior to sequencing [40,44]. The Illumina MiSeq 16S rDNA Microbiome Library (v2; Illumina, San Diego, CA, USA) was constructed and sequenced as per standard company guidelines.…”

The biological effects of microencapsulated organic acids and botanicals induces tissue-specific and dose-dependent changes to the Gallus gallus microbiota

“…The DNA was extracted and sequenced as per standard laboratory guidelines [55]. Brie y, the tissue (ileum or jejunum) contents were thawed, homogenized, and 0.3 g removed followed by extraction using the Qiagen Stool Kit (Qiagen, Hilden, Germany).…”

“…Normalization and library clean-up were also performed prior to sequencing [55,56]. The Illumina MiSeq 16S rDNA Microbiome Library (version 2; Illumina, San Diego, CA, USA) was constructed and sequenced as per standard company guidelines.…”