All animal experimentations were approved by the ethics committee of the Faculty of Veterinary Medicine of the University of Montreal, certificate number 14-Rech-1730. Newly hatched broiler chickens (Ross 308) were purchased at a local hatchery and transported to the avian research center (level 2 confinement facility) of the veterinary medicine faculty. All chickens were vaccinated at the hatchery against Marek’s disease and infectious bronchiolitis. The exact number of analyzed chickens per group per experiment is presented in each table (Tables 1–7). Chickens were placed in two different rooms: the chickens housed in the first room were inoculated with C. jejuni while the chickens housed in the second room were not to be inoculated with C. jejuni. The chickens housed in each room were further separated into two groups: one group received an in-feed supplementation of a selenium-yeast commercial preparation at 0.3 ppm and the other did not. This selenium-yeast concentration is the maximum supplementation allowed by the Canadian Food Inspection Agency (Canadian Food Inspection Agency [CFIA], 2016b). All chickens were fed a standard mash diet and had ad libitum access to water and feed.

At day 12, fresh caecal droppings were collected from each group to confirm the absence of C. jejuni colonization. To differentiate the effect of selenium-yeast from the eventual effect of C. jejuni carriage on the chicken health parameters evaluated, at 14 days of age, one room was inoculated with an oral suspension of two deeply characterized C. jejuni strains (A2008a and G2008b) (Thibodeau et al., 2013, 2015a,b) while the other was not. The oral suspension was obtained from an overnight blood agar culture of each strain that was suspended in PBS phosphate buffered saline (PBS) to an optic density of 1.0 (at 630 nm) and further diluted to obtain a final concentration of 10⁴ CFU per strain per inoculation.

At 35 days of age, chickens were weighed prior to being stunned by electronarcosis and euthanized by bleeding. On each animal, a 10 ml blood sample, a 10 cm segment of the ileum measured from the ileum-caecal junction, as well as the whole caecum were collected. All samples were sent on ice to the laboratory for immediate processing. The in vivo experiment was replicated once more with a distinct lot of birds.

Caecal matter was collected from the caecum. A 1 g portion was used for the enumeration of C. jejuni while another 1 g was flash-frozen in liquid nitrogen and kept at -80°C for DNA extraction (Thibodeau et al., 2015b).

Blood samples were kept 1 h at room temperature and then centrifuged at 100 × g for 15 min. The supernatant was collected and divided into two distinct samples that were kept at -20°C. One sample was used to determine the seric IgY concentrations and the other one was sent to the diagnostic laboratory of the Veterinary medicine faculty for the determination of the total glutathione peroxidase activity (GPX).

The 10 cm ileal segment was opened longitudinally and emptied of its contents with a gloved finger. A sterile microscopic glass slide was then used to scrape off the mucus which was resuspended in 10 ml of cold PBS. The mucus suspension was then kept at -20°C until used for the determination of the intestinal mucus IgA concentration.

The concentration of the total seric IgY and intestinal IgA was assessed by ELISA using commercial kits from Bethyl laboratories (Bethyl Laboratories, Montgomery, AL, USA) for eight chickens per experimental group. Protocols were performed according to the manufacturer recommendations. Serum samples were used at a dilution of 1:50,000 while the intestinal samples were used at a dilution of 1:20. For IgY, the secondary antibody was used at a dilution of 1:20,000 while a dilution of 1:40,000 was used for the IgA.

Total DNA was extracted from all the caecal samples kept at -80°C using a combination of a beads-beating lysis and phenol–chloroform purification as previously described (Thibodeau et al., 2015b). A sample without caecal matter was extracted at the same time as a negative control for use in the downstream molecular biology analysis. DNA concentration was assessed using the Qubit BR assay (Fisher Scientific, Ottawa, ON, Canada). The DNA samples were diluted to a concentration of 10 ng/μl, separated in aliquots, and kept at -20°C until use.

A survey of the chicken caecal microbiota was performed by amplifying and sequencing the V4 region of the 16S rRNA gene from the DNA extracted from the chicken caecal samples, according to Illumina’s “16S Metagenomic Sequencing Library Preparation” guide (Part # 1504423 Rev. B). In each experimental group, the DNA extracted from the ceacum of eight chickens was used. The 16S rRNA gene PCR mastermix (25 μl final volume per reaction) consisted of 1x KAPA HiFi HotStart ReadyMix (Kappa Biosystems, Willington, MA, USA), 600 nM of each primer (Caporaso et al., 2012), 0.4 mg/ml BSA, and 12.5 ng of DNA. The following PCR cycling conditions were used: initial denaturation at 95°C for 5 min followed by 25 cycles consisting of a 30 s denaturation at 95°C, a 30 s annealing at 55°C, and an elongation of 180 s at 72°C that ended with a final elongation of 10 min at 72°C. The amplicons were purified using AMPure XP beads (Beckman Coulter, Brea, CA, USA) according to the manufacturer protocol.

Purified amplicons were barcoded using the Nextera XT Index Kit (Illumina, San Diego, CA, USA) using an eight cycles PCR: initial denaturation at 95°C for 3 min, followed by cycles consisting of a 30 s denaturation at 95°C, a 30 s annealing at 55°C, and an elongation of 30 s at 72°C, and then by a final elongation of 5 min at 72°C. Reactions consisted of 1x KAPA HiFi HotStart ReadyMix (Kappa Biosystems), 2.5 μl of each index, and 5 μl of the purified 16S rRNA gene amplicons. The index PCR was also purified using AMPure XP beads (Beckman Coulter) according to the manufacturer protocol. The purified indexed amplicons were quantified with Qubit HS kit (Fisher Scientific) and diluted to 2 ng/μl. Five microliters of each index PCR was pooled in a single tube and sent to NRC Montréal for MiSeq Sequencing, using the Miseq reagent 500 V2 kit (Illumina) for a 2x 250 bp length, as specified by Illumina.

Raw demultiplexed reads were received from the sequencing center and processed using Mothur version 1.38 (Schloss et al., 2009), following the online MiSeq SOP¹. Prior to OTU clustering, the negative control sample and some samples containing too few or suspicious reads were removed.

To determine OTUs, reads were clustered using Vsearch with the AGC method at the 0.03 level. From this point on, results were analyzed separately for birds raised during the first and second replicate experiment. Reads were aligned and classified using the Silva database (version 123).

For diversity analysis, reads were subsampled or rarefied to the lowest number of reads found in a single sample. The following alpha-diversity indices were computed and compared across conditions: coverage, Sobs, Inverse Simpson, Shannon, and Shannon’s evenness. Beta-diversity analysis was performed by comparing the bird’s microbiota structure using Yue and Clayton diversity index and analyzed by AMOVA and HOMOVA. The composition was analyzed by LDA effect size (LEFSE) (Segata et al., 2011) according to the experimental conditions. The bird’s microbiota compositions were also compared by LEFSE using the phylotype approach. For LEFSE, only significant OTU with a LDA score over 2 were reported. Raw reads for each chicken caecal microbiota analyzed in this study are available through the NCBI SRA database under accession SRP094491.

For all chickens, a 1 g of fresh caecal matter was homogenized in 9 ml of a tryptone-salt solution composed of 0.1% (w/v) tryptone (LabM, Heywood, UK) and 0.85% (w/v) NaCl (Fisher Scientific). For the birds that were not inoculated with C. jejuni, 100 μl of this suspension was plated on mCCDA (LabM) and immediately incubated at 42°C in a microaerobic atmosphere using chemical gas pack generators (Oxoïd, Ottawa, ON, Canada) (Macé et al., 2015). This protocol was also used for confirming the absence of C. jejuni in all birds at day 12.

For the C. jejuni inoculated birds, the caecal suspensions were diluted up to 10⁶ and the last four dilutions were plated on mCCDA (LabM) and immediately incubated at 42°C in a microaerobic atmosphere (Oxoïd). The positive control used for monitoring the adequate C. jejuni growth was C. jejuni strain ATCC 33291. After 48 h of incubation, typical colonies were enumerated and the results were log 10 transformed to assess the effect of selenium-yeast supplementation on the chicken C. jejuni carriage.

To corroborate the colonization status determined by culture, all culture negative and culture positive C. jejuni samples were confirmed by PCR (Yamazaki-Matsune et al., 2007). The PCR mix (25 μl) was composed of primers (C412F at 200 nM, C1228R at 200 nM, C-1 at 800 nM, and C-3 at 800 nM), 1 unit of Taq DNA polymerase (Bio Basic, Markham, ON, Canada), MgSO₄ at 2 mM, and dNTPs at 200 mM. PCR amplicons were visualized on a 1% agarose (Fisher Scientific) gel stained with Sybrsafe (Fisher Scientific). Positive control consisted of DNA extracted from C. jejuni strain ATCC 33291 while the negative control contained no DNA.

Comparison of the chicken’s body weight, C. jejuni colonization levels, seric total GPX activity, seric IgY concentrations, intestinal IgA concentrations, and alpha-diversity indices were analyzed in GraphPad Prism 5 (GraphPad Software, La Jolla, CA, USA). Prior to selecting the correct statistical analysis, the distribution of the data was inspected. When the normality was confirmed, data were analyzed using parametric tests. Otherwise, non-parametric analyses were conducted. An alpha of 0.05 was set to assess significance. Results, with the exception of the microbiota, were analyzed separately for the first or second replicate according to the selenium-yeast or C. jejuni status before being pooled for the analysis of the global effect observed in this study.

After 35 days of growth, no significant difference (p > 0.05) was observed in regards to the chicken final body weight, though the use of selenium-yeast consistently yielded chickens with higher mean body weight values in both replicates (Table 1).

Seric GPX levels were observed to be significantly lower (p < 0.05) for the chickens fed selenium-yeast (Table 2). No significant difference (p > 0.05) was observed for chickens inoculated with C. jejuni, although consistent lower mean values for the inoculated chickens were observed. A large inter-individual variation was also observed, as exemplified by the high standard deviation values.

No significant difference (p > 0.05) was measured regarding total seric IgY concentrations (Table 3). For the intestinal IgA recovered from the ileal mucus layer (Table 4), significantly lower concentrations were observed in selenium-yeast supplemented chickens, but only for the second experimental replicate (p < 0.05). Similarly, IgA concentrations were significantly lower in chickens not inoculated with C. jejuni (p < 0.05), but only for the second experimental replicate.

A total of 8,438,914 sequences were obtained after assembly. Prior to OTU clustering, a total of 4,978,059 sequences remained, representing 308,896 unique sequences. The two negative controls included in this study contained 260 and 427 sequences. Two chicken caecal samples returned numbers of sequences similar to the negative controls (328 and 348) and were discarded from the analysis. Two more samples, originating from chickens not inoculated with C. jejuni, returned unexpectedly high numbers of reads classified as Campylobacter, in similar proportions to all other samples originating from inoculated birds. Further PCR analyses confirmed the samples negative for C. jejuni while all C. jejuni inoculated birds returned a strong PCR signal (data not shown); these samples were removed from the analysis. After this, the lowest number of sequences in a chicken sample was 14,482 while the highest was 161,901.

Alpha-diversity indices (Table 5) were first compared based on the use of selenium-yeast in the feed or the inoculation with C. jejuni. Coverage over 97% was observed in all treatments. In experimental replicate 2, the use of yeast-selenium significantly increased (P < 0.05) the coverage and richness (Sobs) of the bird’s microbiota but this was not observed in experimental replicate 1. Richness (Sobs) was significantly different between the two experimental replicates regardless of the experimental condition (p < 0.001), with mean values of 435 (±130) and 687 (±225) for experimental replicate 1 and 2, respectively. The observed mean Shannon indices (3.50 ± 0.15 and 3.62 ± 0.25 for experimental replicates 1 and 2, respectively) and Shannon Evenness indices (0.58 ± 0.03 and 0.56 ± 0.03 for experimental replicates 1 and 2, respectively) were also significantly different (p = 0.001) between replicates.

The difference in the microbiota structure according to the experimental conditions was investigated. In both replicates, selenium-yeast supplementation (Figure 1) did not influence the microbiota structure, while the inoculation with C. jejuni (Figure 2) did influence the caecum bacterial community. When analyzing all chickens together, the bacterial community structure was significantly different between the experimental replicates (AMOVA p < 0.001, HOMOVA p = 0.2).

The LEFSE analysis identified OTUs that were consistently associated with the inoculation of C. jejuni or the use of selenium-yeast. When using C. jejuni inoculation as a class and selenium-yeast as a subclass, 75 OTUs were identified in replicate 1 and 71 OTUs in replicate 2. Of these, only 17 OTUs were consistently associated in both replicates (Table 6). On the contrary, when using selenium-yeast as the class and the inoculation with C. jejuni as a subclass, for replicate 1, five OTUs were identified compared to two OTUs in replicate 2. For selenium-yeast, no OTU were common to both replicates. All LEFSE results, broken down per replicate, are available in the Supplementary Material.

Using phylotype analysis with genera as the cutoff (mothur taxlevel = 1), 23 OTUs were found to be associated with C. jejuni inoculation for experimental replicates 1 and 2, and the six following OTUs were found for both replicates: unclassified_ Ruminococcaceae, Eisenbergiella, Tyzzerella_3, and [Eubacterium]_hallii_group were associated with the non-inoculated birds while Lachnoclostridium and Campylobacter were associated with the inoculated birds. For supplementation with selenium-yeast, two OTUs were identified in each of the experimental replicates, but none were shared. Apart from the obvious association of sequences from the C. jejuni lineage (Proteobacteria; Epsilonproteobacteria; Campylobacterales; Campylobacteraceae) with C. jejuni inoculated chickens, no other association with treatments were found when using different taxonomical levels (mothur taxlevel 2, 3, 4 or 5) in LEFSE analysis. All LEFSE phylotype results, broken down per replicate, are available in the Supplementary Material.

In experimental replicate 1, the C. jejuni caecal concentrations were found to be slightly but significantly higher for the chicken supplemented with selenium-yeast (Table 7) (p < 0.05). The opposite effect was observed for experimental replicate 2. When all the birds from the two replicates were used together in the same analysis, no significant difference remained (p > 0.05). Confirmation of the presence of C. jejuni in the inoculated chickens was carried out by PCR and culture, which confirmed that all inoculated chickens were infected by C. jejuni while the all the non-inoculated chickens were not.