Reduction in the frequency of Aspergillus spp. in broiler facilities subjected to cleaning and disinfection

ABSTRACT The present study analyzes influence of two cleaning and disinfection protocols on frequency of Aspergillus spp. in broiler facilities. We conducted an observational study, applying two cleaning and disinfection protocols before housing 960 one-day-old broilers randomly allocated in 32 boxes with 30 birds. We considered two types of housing as independent experiments, which differed as to the bedding. First experiment consisted of new bedding materials, which we reused in second experiment. We applied two different treatments, Common treatment included sweeping organic matter, humidifying residual material with low pressure water, washing with water and neutral detergent, with posterior rinsing. European treatment was according to the standards of the manufacturer of disinfectants. Procedures included dry and humid organic matter removal, humidification, washing with high pressure water, application of detergent, rinsing, and application of two compound disinfectants: glutaraldehyde (250 g/L) + formaldehyde (185 g/L); p-chloro-m-cresol (210 g/L). To evaluate the presence of Aspergillus spp., we collected samples from surfaces and equipment before and after cleaning and disinfection, at 7, 14, 28, and 42 days of experimental period. Mycological assessment included streaking on Petri dishes containing AFPA (Aspergillus Flavus and Parasiticus Agar, Sigma Aldrich®). Common cleaning and disinfection treatment led to a higher presence of fungus in floors and walls (83% vs. 0% p = 0.0030 and 50% vs. 0% p = 0.0450) in first and second experiments, respectively. As this study demonstrates by using the European procedures, performing a detailed cleaning and disinfection protocol reduces the presence of Aspergillus spp. in broiler facilities.

at 7, 14, 28, and 42 days of experimental period. Mycological assessment included streaking on Petri dishes containing AFPA (Aspergillus Flavus and Parasiticus Agar, Sigma Aldrich ® ). Common cleaning and disinfection treatment led to a higher presence of fungus in floors and walls (83% vs. 0% p = 0.0030 and 50% vs. 0% p = 0.0450) in first and second experiments, respectively. As this study demonstrates by using the European procedures, performing a detailed cleaning and disinfection protocol reduces the presence of Aspergillus spp. in broiler facilities.

Broiler
facilities have ideal characteristics for the dispersion of fungi and their metabolites (Viegas et al., 2014;2016). Dust particles can act as carriers, facilitating their dispersion and consequent inhalation by birds (Viegas et al., 2015). Bird exposure to Aspergillus spp. spores generally occurs after the introduction of contaminated bedding and feed (Kapetanov et al., 2015). Studies on the presence of Aspergillus spp. in broiler breeders should consider the potential of these fungi to cause economic losses in production. Such losses can occur due to aspergillosis, a respiratory disease with high morbidity and mortality, which can affect all bird species, especially when young (Arné et al., 2011;Kapetanov et al., 2015). Aspergillus spp. also has zoonotic potential, causing pulmonary infections in immunosuppressed populations and allergic reactions in immunocompetent ones. Moreover, Aspergillus spp. dispersion in the rural work environment, especially in poultry facilities, is an occupational health problem (Fairs et al., 2010;Chotirmall et al., 2013;Cafarchia et al., 2014). Procedures for cleaning and disinfecting the facilities, as well as microbiological control of feed, are fundamental for reducing the risk of infections in birds and humans (Grezzi, 2007). These procedures must consider systematic methods. Despite the wide use of water alone for cleaning purposes, studies do not recommend this practice. This also applies to detergents, which although slightly germicidal, are not suitable as disinfectants or sanitizers (Grezzi, 2007;Cafarchia et al., 2014;Kapetanov et al., 2015). Thus, the present study evaluates the influence of two cleaning and disinfection protocols on the frequency of Aspergillus spp. in broiler facilities.

MATERIALS AND METHODS
The present study followed the ethical principles of animal experimentation and was approved by the Ethics Committee on Animal Use of the Faculty of Veterinary Medicine and Animal Science at the University of São Paulo, under Protocol 2029/2010. To evaluate the efficiency in eliminating strains of the fungus Aspergillus spp. in broiler facilities, we conducted an observational study consisting of two cleaning and disinfection protocols before housing 960 one-day-old broilers, which we randomly allocated in 32 boxes with 30 birds each. We raised the birds in these conditions until 42 days of age. We used two types of housing in a conventional shed divided into boxes (2.2 x 1.2 m). These two experiments differed only as to the bedding. The first type of housing included new bedding materials, namely pine wood shavings, which were previously fumigated. The bedding used in the second experiment came from the first experiment and was previously windrowed for anaerobic fermentation. The diet of the animals consisted of corn and soybean meal, being the same for all birds in both housings, meeting the requirements proposed by Rostagno et al. (2011).
We conducted two cleaning and disinfection protocols, applying each of them in 16 experimental boxes of the facilities previously to housing. We applied the protocols to all surfaces and equipment used for broiler breeding, such as: floors, walls, curtains, roofs, screens, feeders, drinkers, operator's boots, bell jars, lamps, shovels, brooms, and buckets. The so-called "common" protocol consisted of simple cleaning and disinfection procedures: sweeping of floors and dry removal of organic matter, wet removal of organic matter with low pressure water, washing with neutral detergent, rinsing, and drying at room temperature. The so-called "European" protocol consisted of detailed cleaning and disinfection procedures, with the concentrations and action times following the manufacturer's guidelines. For that purpose, we performed the following steps: dry removal of organic matter by sweeping; humidification of the environment with low pressure water -application with a hose connected to a conventional tap; washing with water using a jet washer (pressure of 150-180 bar); application of alkaline detergent diluted in water at a concentration of 4%, at low pressure, with 20 to 30 minutes of action; rinsing of the premises with water (pressure of 150-180 bar); drying of the environment; application of disinfectant composed of glutaraldehyde (250 g/L) and formaldehyde (185 g/L) at a concentration of 0.5%, at low pressure, with a backpack sprayer, on all surfaces of the shed and objects; application of disinfectant composed of p-chloro-mcresol (210 g/L) at a concentration of 4%, at low pressure, with a backpack sprayer, only on the floor and walls up to 0.5 m high. To assess the presence of Aspergillus spp., we collected samples before and after cleaning and disinfection, on the 7 th , 14 th , 28 th , and 42 nd days of the experimental period. We analyzed floors, walls, feeders, drinkers, and curtains. For floors, walls, and feeders, we collected three samples per treatment.

RESULTS AND DISCUSSION
The type of bedding used did not influence the results obtained in the present study. The findings corroborate Kapetanov et al. (2015), who studied the presence of Aspergillus in sheds for broilers and turkeys, in beddings and embryonated eggs, using surface swabs. The authors found a 36% frequency for the evaluated surfaces, highlighting the need for sanitary control in reducing the frequency of Aspergillus. Viegas et al. (2011) found Aspergillus in 7% of the air samples and in 5% of the analyzed surfaces. These frequencies are lower than those of the present study; even so, the authors emphasize the importance of identifying Aspergillus in broiler breeding due to the negative consequences to the performance and welfare of the animals (Andreatti Filho, 2000). The wide worldwide distribution of Aspergillus in sheds and hatcheries is demonstrated in the findings of Mustafa (2017), who found high frequencies in Iraq; Lorin et al. (2017), in Romania;and Viegas et al. (2017), in Portugal. Thus, attention should be paid to the health implications from the presence of these fungi in broiler sheds, with the possibility of contamination of animals and development of aspergillosis; contamination of carcasses with mycotoxins; and occupational diseases with the exposure of workers to fungi and mycotoxins (Viegas et al., 2011). The type of ventilation, air humidity, and ambient temperature (Sajid et al., 2006) can influence the occurrence of Aspergillus in poultry facilities. Therefore, it is important to control the microclimate of the facilities so as to reduce the development of these fungi (Cafarchia et al., 2014;Kapetanov et al., 2015). Conventional poultry facilities, as in the present study, have less microclimate control, mainly regarding ventilation, which can favor dispersion and contamination by Aspergillus (Baracho et al., 2001). It is thus fundamental to adopt procedures that reduce the fungal load of the environment, such as cleaning the facilities. Glutaraldehyde and formaldehyde are compounds with good efficiency in eliminating fungi (Peterson et al., 2008) in poultry facilities. These compounds are present in the disinfectants used in the European cleaning and disinfection protocol. Such composition may relate to the lower fungal prevalence in the first and second experiments of the present study. Moreover, the organic matter present in poultry environments acts as a barrier to the action of disinfectants. In this way, procedures such as washing with high pressure water help in its removal, allowing greater efficiency of disinfectants (Peterson et al., 2008). In the common protocol, the absence of an efficient step to remove organic matter may relate to the higher frequencies of Aspergillus.
Cleaning and disinfection procedures such as those performed in the European system are fundamental actions in the prevention of aerial dispersion and contamination by Aspergillus (Rajmani et al., 2011;Cafarchia et al., 2014). Adding to this evidence, the present study demonstrated that performing a simple cleaning and disinfection protocol has little efficiency in reducing the fungal contamination of broiler facilities. The implementation of the European protocol, which consists of sequential practices for the use of detergent and disinfectants with a wide spectrum of action, reached greater efficiency in reducing the frequency of Aspergillus.
Using a cleaning and disinfection program with detailed steps, such as the European protocol, leads to greater efficiency in reducing the occurrence of Aspergillus spp. in broiler facilities.