**5. Formaldehyde fumigation**

Formaldehyde is a byproduct of cellular metabolism and detoxification has been shown to be important for metabolic processes [115]. However, exogenous formaldehyde is a colorless, irritant gas with cytotoxic activity. Due to its solubility in water and biocidal properties, formaldehyde is used as a disinfectant in commercial settings [13]. The first published report of formaldehyde application in commercial hatcheries was in 1908 [9]. For decades, formaldehyde fumigation of hatching eggs has been recommended to control the microbial load in hatching environments [116].

Formaldehyde fumigation has been shown to reduce the bacterial load on the surface of eggshells by 99% [117] and has been used to fog hatching eggs prior to incubation or applied into the hatch cabinet environment during late embryogenesis to control the microbial bloom [6]. The fumigant is typically applied by diffusion of 37% formalin alone or in combination with potassium permanganate inside the cabinet at a single time point or by controlled infusion [118]. Steinlage et al. [118] evaluated the application of 37% formalin applied as a constant rate infusion (CRI, 1 mL/hour over 12 h period) as compared to the traditional method of a single dose application of formaldehyde (12 mL administered at one time point every 12 h). The maximum concentration of formaldehyde in the environment was lower with CRI at 20 ppm versus 102 ppm with the single application of formaldehyde. The effects of each fumigation method on circulating aerobic bacteria in the hatch cabinet, hatchability, and early performance were evaluated and compared to a non-treated control, which received water in lieu of the fumigant In this study, both formaldehyde fumigation methods reduced circulating aerobic bacteria in the hatching environment at DOE20 compared to treatment with water, but the single application of formaldehyde markedly reduced aerobic bacteria in the hatching environment compared to the non-treated and CRI hatchers, and hatchability was improved as a result of formaldehyde fumigation [118]. Although contamination increased because of *in ovo* injection in this study, formaldehyde fumigation reduced the microbial load in the hatching environment and potentially eliminated microbes capable of penetrating eggshells that are lethal to embryonic development. CRI of formaldehyde was effective and likely reduced peak exposure to formaldehyde for neonates and hatchery workers by 10.2-fold. Similar to these results published by Steinlage et al. [118], formaldehyde applied by CRI in commercial hatch cabinets reduced circulating aerobic bacteria 4 h before hatch pull at DOE21 more readily than a single administration of 37% formalin at transfer from incubator to hatch cabinet [119].

Formaldehyde fumigation reduced circulating coliforms in the hatching environment, which reduced horizontal transmission and enteric colonization at hatch [120, 121]. However, formaldehyde fumigation has been associated with tracheal epithelial damage and mucosal sloughing in neonatal chicks [10–12, 122]. At hatch, neonatal chicks are highly susceptible to colonization by respiratory pathogens due to the inherent architecture of the avian respiratory system because the bronchial-associated lymphoid tissue and

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

the immune system do not functionally mature until at least 6 weeks-of-age [123]. The avian respiratory tract has been suspected to be a portal of entry for enteric pathogens, including *S. enterica* [101, 102]. Hence, an insult to the tracheal epithelium, when the neonatal chick is already predisposed to invasion and colonization by respiratory and enteric pathogens, should be avoided.

In 2011, formaldehyde was listed as a known carcinogen by the National Institute of Environmental Health and Safety. In addition to the potential carcinogenic properties of formaldehyde, other negative aspects have been identified [12, 122, 124]. Although the application of formaldehyde during the hatching period effectively reduced aerobic bacterial contamination in commercial hatch cabinets [119, 121], it has been shown that the efficacy of formaldehyde fumigation decreases as contamination increases [125]. Additionally, formaldehyde is not selective and eliminates both beneficial and pathogenic organisms. During late embryogenesis, the fumigant has a limited effect on endogenous microbes inside the egg [117, 120]. The impact of formaldehyde fumigation during late embryogenesis on performance has also been investigated. Zulkifli et al. [122] demonstrated that feed conversion was negatively affected due to formaldehyde exposure. Alternatively, CRI of formaldehyde or a single administration of formaldehyde every 12 h marginally improved feed conversion ratio (FCR) but did not significantly affect body weight gain (BWG) from DOH to day 14 [118]. Mahajan et al. [11] also reported no effects of CRI of formaldehyde on early performance. Contradictory to previous reports, CRI of formaldehyde during late embryogenesis markedly reduced BWG from DOH to day 10 compared to the nontreated control group [124].

Although formaldehyde effectively controls the circulating microbes in the hatching environment, there are no benefits for beneficial pioneer colonization. With the removal of antibiotic growth promoters and the rising concerns regarding antimicrobial resistance, a multifactorial approach to promote early colonization by beneficial microbes and control the microbial bloom in the hatching environment without the use of carcinogenic formaldehyde will be essential.

## **6. Methods to monitor hatchery sanitation**

Controlling pathogens at the hatchery level is critical. Evidence of contamination at the farm level suggests that the hatchery could serve as a primary source of contamination [126]. During the hatching phase, bioaerosols and dust are generated and dispersed by the ventilation system in the hatch cabinet [127]. These bioaerosols circulate in the hatch cabinet, contaminating the environment, equipment surfaces, and fluff, as well as having the potential to affect late embryonic development and neonatal health. To prevent disease transmission and guarantee that disinfection measures are correctly conducted, routine hatchery hygiene monitoring must be implemented. Employee compliance can be improved by using simple microbiological techniques, such as fluff sampling and swabbing of equipment surfaces.

Since the late 1950s, fluff samples have been collected from hatch cabinets to assess the efficacy of sanitization procedures in commercial hatcheries [128]. During the hatching phase, fluff and dander accumulates in the hatching environment and have been shown to contain 4–8 logs of bacteria/g of fluff [81]. Based on the microbial recovery from fluff samples, a rating system was developed to assess the quality of disinfection and fumigation procedures for a particular commercial hatchery [128]. Magwood [129] plated hatcher fluff samples in duplicates both pre

and post-formaldehyde fumigation and applied Wright's rating system. Duplicates were plated to assess the level of variability within a single fluff sample and bacterial and fungal recovery from fluff samples were lower after formaldehyde fumigation. However, both pre- and post-fumigation, the microbial load in the hatcheries with unsatisfactory ratings remained significant [129]. The rating system developed by Wright [128] to assess hatching sanitation practices has been utilized in other investigations [129, 130]. Other investigators also confirmed that fumigation of hatching eggs reduced microbial recovery from fluff collected from the hatch cabinet [131].

The open-agar plate method [119, 121, 132] as well as air sampling machines [133] have been used to evaluate airborne contamination in the commercial hatcheries. For the open-agar plate method, the lid of the petri dish is simply removed, and the agar is exposed to the hatch cabinet environment for a short duration which differs based on the selective nature of the agar media used. Aerosol sampling machines have been investigated as alternatives to the conventional open agar plate method to assess the quality of hatcher sanitation procedures [134, 135]. Gentry [135] sampled various locations in a commercial hatchery using the open-agar plate method and the Anderson air sampler [133] to compare the level of sensitivity for both bacterial and fungal recovery. For a 30 second period, the select environment was sampled using the Anderson air sampler (equated to 0.5 cubic ft) or open agar plates [135]. The Anderson air sampler proved to be the more sensitive method based on overall microbial recovery, specifically using non-selective agar. However, the increased volume of air was sampled with the Anderson sampler versus the inert surface of the agar when using the open-agar plate method, which was reflected by microbial recovery. The volume of air sampled using air sampling machines far exceeded the amount of volume sampled by the open-agar plate method when exposed to the environment for the same duration. These differences must be considered when comparing the two methods as increased time of exposure could negate sensitivity differences.

Magwood and Marr [136] assessed the level of airborne and surface contamination in four commercial hatcheries to determine if aerosol and surface contamination was correlated in a commercial setting. The hatchery environment was sampled to determine airborne contamination, while surfaces in the hatchery, specifically the floors and tables, were swabbed and directly plated on agar media [136]. The authors suggested that direct swabs of select surfaces in the hatchery would be as equally reflective of the level of sanitation as air or fluff samples and was a simpler technique to implement.

The microbial load within the hatch cabinet has been shown to increase with the rise in humidity as chicks or turkey poults begin to hatch [125]. In this study, it was determined that airborne contamination was reflected by eggshell and hatcher surface contamination. Furthermore, it was shown that microbial recovery was lower for hatcheries with adequate sanitation practices while highly contaminated hatcheries had higher microbial loads from hatching cabinet sampling, [125]. These results indicate that horizontal surfaces could be sampled to assess hatchery sanitation procedures implemented to disinfect equipment and control the microbial load in the hatching cabinet. Berrang et al. [132] reported that more salmonellae were recovered from commercial broiler chick hatch cabinets with the open agar plate enrichment method compared to the air sampling machine. However, recovery of Enterobacteriaceae, an indicator of fecal contamination, was increased in samples collected with the air sampling machine compared to the direct open-agar plate method without further enrichment [132]. Thus, sampling method, duration of sampling, sample port location, ventilation system, and type of media used for sampling influence microbial recovery from the hatching environment.

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

In one study, *Salmonella* was recovered from up to 75% of samples collected from commercial hatchery equipment or eggshell fragments recovered from the hatching cabinet [31]. Shell membranes and chick rinses sampling has also been used to assess *Salmonella* Typhimurium contamination in an artificial challenge hatcher model using infected embryonated seeders [100]. In this study, chick rinse samples remained *Salmonella*-negative until the onset of pipping at DOE19. Previous studies have shown that salmonellae are rarely isolated from eggs [137], but the increased percentage of *Salmonella*-positive chicks at hatch suggest moderate replication and dispersion of the pathogen within the hatch cabinet environment. Bailey et al. [138] showed that placement of artificially infected seeder eggs (3 of 200 eggs total, 1.5%) resulted in the colonization of 98% of non-challenged contacts with *Salmonella* at 7 days-of-age. Even though salmonellae presence may appear to be minimal based on microbiological sampling at DOH, infected chicks horizontally transmit the pathogen when comingled with non-infected counterparts [103].

The incidence of *Salmonella* in commercial hatcheries for other gallinaceous species, including geese, has been documented. Chao et al. [139] collected fluff samples, hatch cabinet surface swabs, and shell membranes post-hatch from goose hatcheries and recovered *Salmonella* from ~36% of the fluff samples, 27% from hatch cabinet swabs, and 86% from shell membranes post-hatch. Alternatively, shell membrane samples collected from commercial chicken hatcheries had a significantly lower incidence of *Salmonella* [139]. The authors postulated that the use of formaldehyde in the chicken hatcheries was associated with a greater level of sanitation observed compared to the other poultry hatcheries evaluated. In another study, Zhao et al. [140] isolated *E. coli* from 47 fluff samples collected from commercial hatcheries that contained less virulence-associated genes than the 20 APEC isolates evaluated [140]. However, these samples were collected from formaldehyde-fumigated hatch cabinets and do not provide insight regarding the natural level of contamination in the absence of formaldehyde fumigation.

If hatchery disinfection and sanitation practices are not effective, it will be reflected by hatchability and overall chick quality. Extensive contamination at the hatchery level promotes cross-contamination of strict and opportunistic pathogens during the hatching phase and at the farm. Transmission at the hatchery level can be costly to poultry producers due to reduced performance and potential transmission of foodborne pathogens to consumers. Thus, sampling of the hatching environment (agar plates, aerosol sampling machines, equipment surfaces) and waste generated during the hatching process (fluff, eggshell fragments, post-mortem chick rinses) can provide insight regarding sanitation procedures. These techniques can be utilized to evaluated potential alternatives to formaldehyde fumigation to control the microbial load in the hatching environment.

### **7. Alternatives to formaldehyde fumigation**

Research efforts to identify alternatives to formaldehyde to mitigate pathogen transmission of pathogens in poultry hatcheries have been reviewed [141]. Alternatives to formaldehyde fogging or fumigation of hatch cabinets should have minimal effects on eggshell integrity and hatchability and also inhibit penetration or replication of microbes on the eggshell or within the hatching environment. Eggshell surface contaminants obtained at the breeder facility or during transport should be eliminated prior to incubation to prevent cross-contamination in the hatchery.

Whistler and Sheldon [142] demonstrated that ozone fumigation reduced bacterial growth similar to formaldehyde fumigation when applied for 2 minutes in a prototype setter. Another potential sanitizer, hydrogen peroxide, reduced the microbial load on the surface of the eggshell with minimal effects on structural integrity of the eggshell [2, 143]. Bailey et al. [144] showed that a hydrogen peroxide mist at a concentration of 2.5% limited cross-contamination of *Salmonella* during late embryogenesis compared to UV light and ozone treatment. In this study, the incidence of *Salmonella*-positive eggshells collected at hatch and cecal samples at 7 days-of-age was reduced compared to ozone, UV light, and the challenged control. In a follow up study, efficacy of hydrogen peroxide improved when applied by immersion compared to spray application to the eggshells, but effectiveness was diminished if applied after sufficient *Salmonella* contamination occurred regardless of application method [145]. More recently, application of 30% hydrogen peroxide by vaporization reduced total aerobic bacterial recovery from the eggshell and did not impact hatchability or early performance [146]. Thus, contamination prior to treatment should be limited. Additionally, aerosolized application of sanitizers would be more feasible than immersion in commercial hatchery operations.

Eggshell surface contamination was reduced after application of hydrogen peroxide in conjunction with UV light exposure, referred to as an Advanced Oxidation Process [147, 148]. The combined treatment only reduced the incidence of *Salmonella* on the surface of the eggshell, and did not prevent bacterial penetration of the eggshell [147]. The incidence of *Salmonella* in the GIT of chicks and early performance were not reported in this study. However, Rehkopf et al. [149] showed that UV light exposure and hydrogen peroxide treatment to eggshell surfaces prior to incubation reduced *Salmonella* enteric colonization at DOH and at 14 days-of-age. More recently, Melo [150] evaluated UV irradiation, ozone fumigation, hydrogen peroxide spray, or peracetic acid spray as potential alternatives to paraformaldehyde fumigation for hatching eggs. UV treatment and spray application of peracetic acid more effectively reduced total aerobic bacteria on eggshells compared to all treatment groups, including formaldehyde [150]. However, both UV and peracetic acid treatment actually increased total aerobic bacteria and Enterobacteriaceae recovered from yolk samples 24 h post-hatch as compared to non-treated controls and formaldehyde treated group [150]. Another alternative sanitizer, chlorine dioxide was applied at a concentration of 0.3% to hatching eggs at 18 days of embryogenesis but did not effectively reduce the microbial load on the eggshell compared to formaldehyde and had no effect on performance [11]. Introduction of an artificial challenge and additional sampling would provide more insight as to the effectiveness of candidate disinfectants.

Some additional naturally-derived candidates have also been evaluated. Eggshells were treated by spray application of grain alcohol, clove essential oil, or an ethanolic extract of propolis, a component of bee hives, and compared to sanitizing eggshell with paraformaldehyde prior to incubation [151]. In this study, application of the ethanolic extract of propolis negatively impacted hatchability of fertile eggs and significantly increased late embryonic mortality compared to the other treatment groups, which was likely associated with impaired gas exchange and moisture loss during incubation. Similar to paraformaldehyde fumigation, spray application of clove essential oil eliminated Enterobacteriaceae on the eggshell surface and had no apparent effect on integrity of the eggshell [151, 152]. Pyrazines are naturally-occurring organic nitrogen-containing ring structures which can be chemically synthesized or obtained by microbial fermentation [153]. Alkyl pyrazines are typically used as flavoring agents or as fragrances) and have been shown to have antimicrobial activity [154].

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

Application of a volatile organic compound, an alkylated pyrazine (5-isobutyl-2,3-dimethylpyrazine), reduced viable microbes on the surface of the eggshell [155]. However, since overall eggshell contamination was low and the effects of the treatment on eggshell quality and chick viability were not assessed, future studies are required to validate efficacy and feasibility of alkylated pyrazine.

The effect of spray application of probiotics into commercial hatch cabinets as a potential replacement for formaldehyde fumigation has also been preliminarily investigated. Although the Gram-negative bacterial bloom was elevated in probiotictreated hatchers, probiotic application effectively reduced GIT coliforms of neonatal chicks compared to chicks placed in formaldehyde fumigated hatch cabinets [121]. Compared to formaldehyde fumigation, probiotic-application would not be expected to inhibit the microbial bloom in the hatching environment, but the beneficial microbes could perhaps displace the opportunistic pathogens in the hatching environment thereby promoting colonization by beneficial microbes.

In future studies, the ability of candidate alternatives should be evaluated under artificial challenged conditions to assess the impact on microbial load in the hatching environment and enteric colonization at hatch. Sampling the environment in the hatch cabinet during the hatching phase would provide insight on the microbial load compared to traditional formaldehyde fumigation. Furthermore, eggshell quality may be compromised due to treatment and have detrimental effects on embryonic development and should be evaluated. Although chemically and naturally-derived sanitizers reduced the microbial load on the eggshell and potentially limited horizontal transmission of pathogens in the hatchery setting, these compounds lack the ability to competitively exclude pathogens. Since formaldehyde non-selectively acts on microorganisms on surfaces or in the environment eliminating both beneficial and pathogenic microbes, artificial introduction of probiotic candidates during the hatching phase may be a promising method to enhance enteric colonization by beneficial microbes.

### **8. Conclusion**

Formaldehyde effectively controls the microbial load on the surface of eggshells and in the environment, but identification of alternatives to formaldehyde represent an opportunity for improving the health and performance of postnatal chicks. Exposure to opportunistic pathogens during the neonatal period can be costly to poultry producers and reduction of infection and impact remains a worthy goal. Since the level of natural contamination is inherently variable, reproducible laboratory challenge models are essential for development and validation of alternatives to formaldehyde fumigation to control the microbial load in commercial hatch cabinets. Artificial challenge models to simulate exposure to hatchery-relevant pathogens during the neonatal period have been employed, including direct application of the challenge to eggshells (spray, immersion, etc.), *in ovo* application, and horizontal transmission models. Additionally, prophylactic use of antibiotics in the feed has previously been used to control bacterial infections and improve growth performance. Emergence of multi-drug resistant strains of bacteria and concern for human health has limited the use of antibiotics in commercial poultry production. Thus, a multifaceted approach to control the microbial bloom in the hatching environment and promote pioneer colonization by beneficial organisms that is applicable to the poultry industry is a major unmet opportunity.

*Broiler Industry*
