**4. Opportunistic pathogens associated with commercial poultry hatcheries**

In integrated poultry production systems, transfer of the maternal microbiota is limited. Commercially reared chicks are exposed to the plethora of environmental microbes in the hatchery. Cleaning and disinfection processes are implemented to control the microbial bloom in the hatchery, such as formaldehyde fumigation. Environmental contamination dictates the pioneer colonizers of the gastrointestinal tract, influences performance, and resistance to opportunistic pathogens throughout the life of the animal.

The composition of the microbial bloom can be impacted by placement of contaminated non-viable embryonated eggs in commercial hatch cabinets. As non-viable embryonated eggs incubate, the internal pressure increases within the egg and may rupture or explode. In doing so, the surface of viable embryonated eggs in proximity is contaminated with non-viable embryonated egg material, which also influences the level of environmental contamination that occurs during the hatching phase. Non-viable embryonated eggs have been shown to be predominantly contaminated with *Micrococcus* spp. and Enterobacteriaceae and the level of contamination directly affected embryonic development [80]. Moreover, at DOE21, bacteria recovered from non-viable embryonated eggs was ~2.4 logs higher than the chicks that successfully hatch [81]. In a more recent study, *Enterococcus faecalis* was shown to be the most

abundant *Enterococcus* spp. recovered from non-viable embryonated eggs, while 56% of the non-viable embryonated eggs contained both *E. faecalis* and *E. coli* [82]. Additionally, Karunarathna et al. [83] demonstrated that non-viable embryonated eggs are potential reservoirs for enterococci and *E. coli*. In this study, antimicrobial resistance phenotypes were observed for up to 40% *E. faecalis* isolates and 37% of the *E. coli* isolates recovered from non-viable embryonated eggs [83]. Both *E. coli* and *E. faecalis* are a part of the commensal microflora, but co-infection with avian pathogenic *E. coli* (APEC) and *E. faecalis* may be associated with increased colibacillosis-related mortality in both chickens and turkeys [84]. Recovery from the yolk sac suggests that the navel is a critical portal of entry for *E. faecalis* during the neonatal period [84]. Reynolds and Loy [85] isolated *E. faecalis* from game birds in the United States. The ring-neck pheasant eggshells and embryos harbored pathogenic *E. faecalis* that have been shown to negatively impact hatchability [85]. Transmission of opportunistic pathogens, including *E. faecalis* may occur via horizontal or vertical transmission. The inherent risk of vertical transmission of *E. faecalis* from broiler breeders to broiler chicks increased as the breeder hens aged (>42 weeks of age) which promoted horizontal transmission of *E. faecalis* during the hatching phase [86]. Moreover, antimicrobial-resistant *E. faecalis* strains have been isolated from broiler breeder hens [87]. Thus, potentially pathogenic and antimicrobial-resistant *E. faecalis* may be vertically transmitted from breeder hens to progeny and subsequently horizontally transmitted to naïve chicks at hatch.

Methods to prevent vertical transmission of APEC from breeder hens to offspring are essential to prevent horizontal transmission at the hatchery level [88]. Portals of entry of APEC include the respiratory tract or translocation from the intestinal tract during stress [89]. APEC strains cause primary and secondary extra-intestinal infections, however, successful colonization of the air sacs by APEC subsequently leads to a systemic infection. APEC strains contain virulence factors and proteins that promote adherence and colonization of that respiratory mucosa and air sacs [90] by evading host immune defenses [91]. Embryonic infection by APEC may or may not be lethal to a developing embryo. For instance, to evaluate vertical transmission of APEC, Giovanardi et al. [92] isolated APEC from two broiler breeder flocks and their progeny. The APEC strains isolated from the breeders and progeny were genetically similar, which signifies the importance of APEC control at the breeder level [92]. APEC infection has also been associated with increased 7-day mortality related to airsacculitis and colisepticemia [93]. Horizontal transmission of APEC during late embryogenesis has been replicated in small-scale hatch cabinets [94, 95]. Exposure to APEC post-lay or during embryogenesis may not always impact hatchability, but colonized chicks can serve as seeders to horizontally transmit the pathogen during the hatching process or production period.

Although *E. coli* and *E. faecalis* are frequently isolated from neonates, other presumptive pathogens must be considered. *Staphylococcus aureus* contamination in hatcheries has been shown to increases morbidity and mortality in chickens [96]. There is evidence of *S. aureus* jumping from humans to poultry approximately 38 years ago due to an adaptation to increased resistance to host heterophils [97]. In 2009, *S. aureus* isolates recovered from poultry were predominantly related to a clonal complex relevant to humans [97]. Although *S. aureus* was not typically associated with disease in poultry ~50 years ago, there has been pressure to adapt, thus leading to the emergence of *S. aureus*-associated diseases in poultry. Mobile genetic elements (MGEs) facilitate horizontal gene transfer and were identified in the *S. aureus* recovered from poultry sources, but were not present in the *S. aureus* strains recovered

*Value and Limitations of Formaldehyde for Hatch Cabinet Applications: The Search… DOI: http://dx.doi.org/10.5772/intechopen.104826*

from humans [97]. Perhaps the unique MGEs are responsible for the host-specific pathogenesis of select *S. aureus* strains affecting commercial poultry. Additionally, severe *S. aureus* contamination in the hatchery may induce pneumonia further validating the need for control at the hatchery level [98]. Other investigators have also speculated that *S. aureus* on the hands of hatchery and parent flock personnel may contribute to increased *S. aureus*-associated skeletal diseases in broiler chickens [99].

Neonatal broiler chicks are far more susceptible to *Salmonella* colonization, with susceptibility decreasing as the GIT microflora mature. The first critical point for horizontal transmission of *Salmonella* to occur is at the hatchery level. As previously mentioned, *Salmonella* spp. readily penetrate the eggshell [51]. Successful eggshell penetration by *Salmonella* does not necessarily have to occur during embryogenesis. For example, Cason et al. [100] demonstrated that initial *Salmonella* recovery from yolk sacs, GIT, and chick rinses remained low until the onset of pipping [100]. This suggests that oral ingestion of the bacterium during the pipping process was sufficient enough to cause infection. Although the oral route has been thought to be the primary route of infection for *Salmonella*, evidence suggests that the respiratory route should be considered as a viable portal of entry for *Salmonella* [101, 102]. This is critical because bioaerosols are generated throughout production in commercial poultry operations. Cason et al. [1] demonstrated that horizontal transmission of *Salmonella* occurs during the hatching phase by comingling seeders embryos, or embryos directly inoculated with *Salmonella* at DOE18, with non-challenged, naïve embryos in a hatch cabinet. *Salmonella* was recovered from air samples collected from the hatcher environment and the GIT of non-challenged contact chicks at hatch [1]. Cross-contamination may also occur during the post-hatch phase during handling, transport, and placement at the farm. For example, in one study, infecting 5% of the population with 102 CFU of *Salmonella* Typhimurium (seeders/sentinels) at hatch was sufficient to contaminate 56.7% of the non-infected counterparts within the same pen [103]. This suggests that low-level *Salmonella* contamination at the hatchery level may increase the risk of horizontal transmission at the flock level. Furthermore, salmonellae have evolved mechanisms to evade host defenses to establish colonization and promote tolerance [104]. In the absence of stress, the infection can persist in asymptomatic carriers and remain undetectable. Although susceptibility to *Salmonella* infection decreases with age, stressful events, such as feed withdrawal, promote litter pecking and coprophagic behavior, increasing the prevalence of *Salmonella* in the crop of broiler chickens at processing [105]. Thus, it is imperative to limit horizontal transmission of *Salmonella* during the neonatal period.

Fungal contaminants, such as *Aspergillus* spp. are ubiquitous in commercial poultry hatcheries [106–108]. *Aspergillus fumigatus* is the most common cause of aspergillosis in poultry [109]. A single *Aspergillus fumigatus* hyphae produces thousands of hydrophobic conidia (spores) that are readily dispersed into the environment [109]. Inhalation of *Aspergillus fumigatus* spores has been associated with respiratory mycosis, or brooder pneumonia [6, 110]. These fungi degrade the cuticle of the eggshell and increase the likelihood of invasion during embryogenesis [43, 111]. Application of *Aspergillus fumigatus* spores in a wet suspension or dry suspension increased embryo contamination and incidence of aspergillosis [112]. Huhtanen and Pensack [113] showed that washing eggs with water contaminated with *Aspergillus fumigatus* spores prior incubation markedly reduced hatchability. Moreover, *Aspergillus fumigatus* conidia can replicate in the air cell, which is inaccessible to any fungicidal compounds applied during the hatching phase [114]. The egg yolk in non-viable embryonated eggs also serves as a nutritive source for *Aspergillus fumigatus* [114].

The 21-day embryonic period makes up 28% of the entire lifespan of a modern commercial 52-day-old broiler chicken. It is important to limit transmission of opportunistic pathogens during embryogenesis. Although the microbial bloom during the hatching phase has been controlled with formaldehyde, efficacious alternatives to formaldehyde are needed that favor colonization by beneficial microbes and improve poultry health.
