**3. Pioneer colonization of the gastrointestinal tract (GIT): critical timepoints during the neonatal period**

Pioneer or initial colonizers of the neonatal GIT influence the diversity of the post-hatch intestinal microbiome [17, 18], promote functional development of the immune system [19], and inhibit colonization by enteropathogenic bacteria [20]. Once established, the commensal microbiota inhibits pathogen invasion and colonization by forming a microbial barrier and by competing for nutrients and attachment sites [21]. The commensal microbiota also modulates host immune development and maturation of the GIT [19]. The intestinal immune repertoire evolves to tolerate the resident microbes in the lumen of the GIT, which is critical for homeostasis [22]. Pioneer colonization of the neonatal intestinal tract occurs at birth (mammalian species) or hatch (avian species). For mammalian species, transfer of the maternal microbiota to progeny occurs during vaginal birth where the composition of the neonate's intestinal microbiota tends to resemble the vaginal microbiota [23]. For avian species, transfer of the maternal microbiota occurs during oviposition [24] and post-hatch due to coprophagic behavior or cloacal sampling of the nest or maternal environment. Cloacal sampling and uptake by retrograde transport of environmental antigens to the bursa of Fabricius has been shown to stimulate immune development [25, 26]. Perhaps coprophagy and cloacal drinking amplify antigen exposure during the neonatal period before maternal immunity wanes. Additionally, cloacal drinking is known to transmit organisms directly to the ceca along with retrograde urine transport [27–29] and intracloacal administration of beneficial bacteria has been shown to be markedly more potent than oral administration with regard to exclusion of selected cecal pathogens [30, 31].

During incubation of eggs by hens, it has been shown that the number of pathogenic microbes on the eggshell decline during incubation, and resident microbes on the eggshell inhibit trans-shell invasion by pathogens [32, 33]. However, in commercial poultry operations, embryonated eggs immediately removed from the hen may be exposed to fecal or environmental microbes that adhere to and potentially penetrate the eggshell [1, 34]. The risk of trans-shell invasion appears to be relative to the amount of contamination in the environment at the time of oviposition. Smeltzer et al. [14] observed that floor eggs had more contamination and greater susceptibility to bacterial penetration than nested eggs. The increased contamination was likely associated with increased fecal debris on the surface of the eggshell of floor eggs. Preventing transmission of pathogens during the perinatal and postnatal periods is critical to improving poultry health and optimizing performance. For instance, early colonization by beneficial microbes during late embryonic development improved growth performance and immune system development [35, 36]. However, enteric pathogens, including *Salmonella enterica* serovar Typhimurium, capitalize on the host's inflammatory response to alter the composition of the commensal microbiota to enhance colonization of the enteropathogen [37, 38]. Moreover, the energetic costs

related to the activation of inflammatory pathways by opportunistic pathogens have been shown to cause protein catabolism [39]. Thus, it is important to mitigate exposure to and transmission of pathogenic microbes in the hatchery to optimize poultry health and performance, but at present, mitigation efforts also destroy some eggshell defenses and reduce the opportunity for beneficial pioneer colonization.
