Initial intestinal microbial colonization is thought to have a major influence on early animal health. The commercial practice of hatcheries and delaying microbial colonization by pioneer colonizers may influence unwanted pathogenic bacterial growth, altering the development and maturation of the GIT. Recent studies of gut microbial establishment currently suggest that the initial inoculation and colonization of the chicken GIT microbiota can have a major influence on the growth performance and health of birds. However, the impact of different types of pioneer colonizers on the development of the microbiome community and GIT are not well-studied, especially direct comparison of Enterobacteriaceae and lactic acid producing (LAB) strains. Although Enterobacteriaceae are found in the neonates of mammals and avians alike, the role of Enterobacteriaceae as apathogenic genera in relation to development of the avaian microbial community and intestinal physiological development reains elusive. The first objective (Chapter 3) was to compare total Enterobacteriaceae and LAB recovery with culture techniques to total reads to corresponding operational taxonomic unit (OTU) Enterobacteriaceae and LAB generated from Illumina MiSeq sequencing from matched chick cecal samples at three and ten days post hatch. Results showed at three days the Pearson’s correlation value of -0.082 between CFU of culturable Enterobacteriaceae and correspoinding OTU reads and culturable LAB CFU and correspoinding OTU reads had a correlation of 0.097. The best fitted model, linear model resulted in p-values of 0.606 and 0.551 for Enterobacteriaceae and LAB respectively, further illustrated the lack of correlation between reads to CFU/g ceca recovery. By ten days of age the relative abundance of CFU and the number of reads were numerically similar with 8.1%, 4.6% Enterobacteriaceae counts and 91.9%, 95.4% of LAB counts, respectively. However, no correlation of OTU and CFU occurred on counts or culturable Enterobacteriaceae (¿=-0.049; p-value = 0.769) and LAB (¿= 0.290; p-value= 0.066) at 10 days. The CFU may be appropriate to identify a few families or orders of culturable bacteria that change because of a treatment or product. Without identifying viability cells to the DNA recovered from NGS, there will always be the question whether the OTU counts in the intestinal tract of any animal is accurate.
The objective of the second study (Chapter 4) was to evaluate the intestinal microbial changes of the chick at day-of-hatch (DOH) through 10 days of age in relation to different pioneer colonizers, apathogenic Gram-negative isolates and a LAB probiotic candidate. At embryo day (ED) 18, embryos were inoculated with either saline (S), or ~ 102 CFU of Citrobacter (C), Citrobacter 2 (C2) or a LAB-probiotic (L) in the amnion. The whole-GIT, at DOH and the ileum and ceca, at 10d were collected. Once the DNA was isolated from mucosal and digesta contents, samples underwent 2 x 300 paired-end Ilumina MiSeq library preparation, targeting the V4-V5 region of the 16S rRNA gene for microbiome analysis. An increased abundance of Lactobacillaceae family and Lactobacillus genus were observed in the L group at DOH (p < 0.05). While Lactobacillus salivarius was one of the pioneer colonizers in the GIT at ED18, the population decreased by 10 days of age (39.59% to 0.09%) and replaced with a population of undefined Lactobacillus genera (10.36%) and Lactobacillus returi (3.63%). The L treatment may be excellerating into a mature microbiota much earlier as obligate anerobe Clostridium piliforme was only populated in L DOH chicks (1.58%). While Enterobacteriaceae was the dominant family in C group at DOH (p < 0.05), primarily from the Citrobacter isolate inoculated in ovo (57.44%) by 10 days, Enterobacteriaceae populations decreased and replaced completely by E.coli (0.06%). Administrating another Citrobacter strain in ovo (C2) did not dominate the GIT of DOH chicks (7.92%), but had the highest overall abundance of undefined Lactobacillus in the ceca by 10 days of age (25.28%).
The final objective (Chapter 5) was to identify developmental changes, measured as the GIT proteome, of day of hatch chicks in relation to the same pioneer colonizers in Chapter 4. A total of 493 proteins were identified for differential comparison to S at a significance level of p=0.05. Different levels were noted in 108, 39, and 78 proteins in C, C2, and L groups, respectively, which were uploaded to Ingenuity Pathway Analysis to determine associated canonical pathways and biological functions related to these changes. Predicted upstream regulators in the cytokine family up-regulated by C2, indicated with a z-score =1.000, included IL1ß, IL6, and OSM which suggested an overall pro-inflammatory condition of the GIT. This was consistent with one of the canonical pathways up-regulated exclusively in C2, acute phase response signaling (z = 2.000, p = 8.13E-04). This was in contrast to a down-regulation, z-score =1.000 or no change to TNFa, IL-4, IL-15 and IFN¿ pro-inflammatory cytokines and up-regulation (z = 2.000) of anti-inflammatory IL-13 in L treated groups,. This would indicate a state of decreased inflammatory status within the GIT. This was further illustrated by increased activity (z = 1.000) of 96 molecules associated with cellular assembly, organization, function, signaling and maintenance in C2 infected samples, versus overall down-regulation in L treated samples, which could be associated with cellular injury and repair. The down-regulation of canonical pathways actin cytoskeletal signaling and integrin signaling (z = -1.000, -1.342, p < 1.00E-03) in L further supports the decreased cellular damage. Leukocyte extravasation signaling (z =1.000, p = 0.017), and NRF2-mediated oxidative stress response (z = 2.000, p = 2.86E-04) were up-regulated in C. While C may also be in a dis-regulated inflammatory state (up-regulation of NFkB, IgG, OSM, TGFß1), the impact of pathways differ from C2.
Taken together, providing different isolates in ovo can have a strong impact on initial colonization of the provided isolate by DOH. Ultimately, pioneer colonizers strain selection is key as two different Citrobacter strains impacted the microbial environment differently, and the DOH intestinal status were unique yet likely undesierable. Providing a LAB-probiotic may increase colonization of desirable bacterial groups in the ceca by 10 days of age and the DOH intestinal status provides groups of proteins involved in the structural development and immune status that may be manipulated by different probiotic candidates.