One Health Approach to Tackle Microbial Contamination on Poultries—A Systematic Review

This study reports the search of available data published regarding microbial occupational exposure assessment in poultries, following the PRISMA methodology. Air collection through filtration was the most frequently used. The most commonly used passive sampling method was material collection such as dust, cages, soils, sediment, and wastewater. Regarding assays applied, the majority of studies comprised culture-based methods, but molecular tools were also frequently used. Screening for antimicrobial susceptibility was performed only for bacteria; cytotoxicity, virological and serological assays were also performed. Most of the selected studies focused on bacteria, although fungi, endotoxins, and β-glucans were also assessed. The only study concerning fungi and mycotoxins reported the carcinogenic mycotoxin AFB1. This study gives a comprehensive overview of microbial contamination in the poultry industry, emphasizing this setting as a potential reservoir of microbial pathogens threatening human, animal, and environmental health. Additionally, this research helps to provide a sampling and analysis protocol proposal to evaluate the microbiological contamination in these facilities. Few articles were found reporting fungal contamination in poultry farms worldwide. In addition, information concerning fungal resistance profile and mycotoxin contamination remain scarce. Overall, a One Health approach should be incorporated in exposure assessments and the knowledge gaps identified in this paper should be addressed in further research.


Introduction
The One Health approach incorporates human, animal, and plant health, as well as the health of their shared environment, for supporting a multidisciplinary and holistic approach that integrates monitoring, planning, and evaluation to optimize co-benefits and public health outcomes [1,2]. In addition, the One Health approach supports global health by fostering coordination, collaboration, and communication among different sectors at the human-animal-environment interface to address common health threats such as antimicrobial resistance (AMR), food safety, zoonotic diseases, and several others [2,3].
The industrialization of the poultry sector poses a considerable negative impact on air, soil, and water. The increase in waste management problems can be considered as one of the major drivers fostering harmful effects on environmental health [4]. Indeed, pathogens can be disseminated by unrecognized pathways, for example, on airborne dust Table 1. Inclusion and exclusion criteria for the articles selected.

Inclusion Criteria Exclusion Criteria
Articles published in the English language Articles published in other languages Articles published from 1 January 2000 to 20 January 2023 Articles published prior to 2000 Articles reporting findings from any country Articles related to biocontrol efficacy or related to clinical trials Articles related to microbial exposure from poultries and related products Articles related to biocontrol efficacy or without mention of microbial exposure or metabolites Original scientific articles on the topic Abstracts of congresses, reports, reviews/state of the art articles

Study Selection and Data Extraction
The selection of the articles was performed through the Rayyan intelligent systematic review application, which is a free web-tool that greatly speeds up the process of screening and selecting papers for academics working on systematic reviews, in three rounds.
The first round was conducted by one investigator (BG) and comprised the screening of all titles to eliminate papers that were duplicated or unrelated to the subject.
Rayyan was then used to analyze the papers that were chosen. The second round was a screening of all abstracts carried out by two investigators (BG and RC). The full texts of all potentially relevant studies were evaluated in the third round, taking into account the inclusion and exclusion criteria. Potential divergences in the selection of the studies were analyzed and resolved by four investigators (BG, MD, RC, and PP). Data extraction was then conducted by BG. It was also checked over by MD and CV. The following details were manually extracted: (1) databases, (2) title, (3) country, (4) environment assessed, (5) objective, (6) microorganisms and metabolites, (7) analyzed matrices, (8) sampling methods, (9) analytical methods, (10) main findings, (11) references.

Quality Assessment
The assessment of the risk of bias was performed by two investigators (BG and CV). Within each study, we evaluated the risk of bias across three parameters divided into Each parameter's risk of bias was rated as "low," "medium," "high," or "not applicable." The studies for which all the key criteria and most of the other criteria were characterized as "high" were excluded.

Results
The workflow diagram for selecting studies is illustrated in Figure 1. Initially, 259 studies were found in the database search, from which 197 abstracts were examined and 97 complete texts were assessed for eligibility. After considering the inclusion and exclusion criteria, a total of 39 studies were disregarded, mostly because they were related to biocontrol efficacy, clinical trials, or biological samples. A total of 58 studies related to microbial exposure in poultry facilities were selected.

Quality Assessment
The assessment of the risk of bias was performed by two investigators (BG and CV). Within each study, we evaluated the risk of bias across three parameters divided into key criteria (environment assessed, microorganisms and metabolites, sampling methods, analytical methods).
Each parameter's risk of bias was rated as "low," "medium," "high," or "not applicable." The studies for which all the key criteria and most of the other criteria were characterized as "high" were excluded.

Characteristics of the Selected Studies
[59]     In chicken farms, a total of 22 Gram-positive bacterial species, three Gram-negative bacterial species, and five fungal species were identified. All broiler farms exceeded the recommended stocking density (0.066 m2/head), which may have led to the higher endotoxin concentrations in indoor dust from chicken farms than pig or cattle farms. Monitoring the indoor airborne endotoxin level was also found to be critical for risk assessment of health for animals or workers.
[61]  Detailed 16S rRNA gene sequence analyses showed potential exposure to risk group 2 bacteria at the hatchery workplace. A size fractionated sampling device revealed that pathogenic bacteria would reach the inhalable, thorax, and possibly alveolar fraction of lungs.
Culture-based methods Molecular tools (16s rRNA gene sequencing) More than 50% of bacterial isolates were phylogenetically most closely related to bacterial species of risk group 2. There were high concentrations of risk group 2 bacteria, which have been implicated in different human respiratory disorders. Adequate breathing protection for employees is recommended during sorting of ducklings. [28]

Culture-based methods
Among poultry buildings, the broiler house showed the highest exposure level and emission rate of total airborne bacteria and fungi, followed by the layer house with manure belt and the caged layer house. The highest exposure level and emission rate of airborne microorganisms found in the broiler house could be attributed to sawdust, which can be dispersed into the air by the movement of the poultry when it is utilized as bedding material.
[60] The microbial flora was dominated by Gram-negative and coagulase-negative staphylococci for bacteria and by species belonging to Cladosporium, Penicillium, and Aspergillus genera for molds. Overall, microbial levels were below the occupational limits. However, the microorganisms identified may exert adverse effects on exposed workers, in particular for those engaged in the early slaughtering stages, as evidenced by the presence of pathogenic species. Additionally, the detection of pathogenic bacteria near air handling units may constitute a risk to public health and of environmental pollution.
[42]  Campylobacter jejuni isolates were resistant to tetracycline, erythromycin, azithromycin, and clindamycin. Some organic chicken lots sampled in Quebec were positive for C. jejuni, which establishes this presence for the first time and suggests a possible contribution of these types of production to human campylobacteriosis. [71] Risk characterization of antimicrobial resistance of Salmonella in meat products Spain Animal farms (poultry, pork, and beef farms; 95% industry and 5% retail)

Food safety Bacteria
Animal carcasses (fresh poultry, n = 234); pork, n = 196); beef, n = 29; minced poultry, n = 151; pork, n = 1270; and beef, n = 170) Passive methods (material collection) Culture-based methods Antimicrobial susceptibility Salmonella isolates found in poultry had a high level of resistance to nalidixic acid, while those found in pork were more resistant to tetracycline and ampicillin. Furthermore, 41% of Salmonella isolates were resistant to three or more antibiotics. Additionally, risk characterization was estimated.
As a result, three cases were classified as "very high additional risk," all of them in minced meat, two cases in poultry (gentamicin and nalidixic acid), and one in pork (ampicillin). [45] The median concentrations of the endotoxin in dust determined with LAL tests in sheep sheds, poultry houses, and horse stables were 15,687.5 µg/g, 8081.8 µg/g, and 79.3 µg/g, respectively, while those determined with the GC-MSMS technique were 868.0 µg/g, 580.0 µg/g, and 496.0 µg/g, respectively. In conclusion, endotoxin in the concentrations detected in this study may present a respiratory hazard to both livestock animals and farm workers.
[33]  were recovered from live incoming birds, equipment, and processed carcasses in the two processing plants. Indeed, forty-six (36.5%) of the isolates were resistant to six or more of the antimicrobial agents tested.
[75] A large fraction (up to 37%) of particles from 2-10 µm was found to be fungal spores. Each type of agricultural environment was found to have specific characteristics of exposure. Harvesting was dominated by exposure to large dust particles with a large fraction of fungal spores, whereas the particle size distributions in animal confinements were dominated by small particles.
[65] The farm dust resistome contained a large variety of ARGs; more than the animal fecal resistome. The farm dust resistome from European poultry and pig farms is equally or more abundant and rich than the resistome of poultry and pig feces and farmers. A positive association between on-farm antimicrobial usage in animals on the farm and the total abundance of the dust resistome was found. Briefly, poultry and pig farm dust resistomes are rich and abundant and associated with the fecal resistome of the animals and the dust bacterial microbiome [51] Fluoroquinoloneresistant Escherichia coli isolated from healthy broiler s with previous exposure to fluoroquinolones: Is there a link?

Iran
Poultry farms (n = 7) Human health due to environmental impact Bacteria Samples from broiler chickens and turkeys previously exposed to both quinolone (flumequine) and fluoroquinolone (n = 95) UK Culture-based methods Antimicrobial susceptibility The differences between ciprofloxacin resistance rates in strains from chickens with previous exposure to fluoroquinolones compared with isolates from chickens without a history of drug use were significant (49.5% vs. 33.7%, p =/0.0461). It seems that use of fluoroquinolones constitutes a major selective pressure for resistance.
[63] Culture-based methods Antimicrobial susceptibility Individual birds within each of the flocks involved in the current study were 70 to 100% colonized prior to loading and transport. Levels of Campylobacter spp. found in production and in processing were not strongly correlative, indicating the existence of complex parameters involving production factors and variables associated with flock transport and the processing of the broilers. The sources of Campylobacter sp.
appear to be diverse, and discussion regarding the optimum approach for the control of the organism during poultry production remains lively. [69] A

Culture-based methods
There was a positive association between the quantity of Gram-negative bacteria in the litter in the front third of the house (the brooding area) during the brooding period and the percentage of cellulitis. [68]

Culture-based methods
The most common airborne fungi, inside the poultry house, as well as in the surrounding areas, were Penicilium sp., Aspergillus sp., Cladosporium sp., and Alternaria sp. The majority of the identified fungal species were characterized as potential allergens and exposure to their spores may provoke immune response in susceptible individuals.
[32] 94b The concentration of fungal aerosols in the poultry houses increased as the ages of the broilers increased, which was also accompanied by gradual increases in the variety and diversity indices of the fungal communities in the air of the poultry houses. Overall, the dominant fungal genera found may be harmful to the health of poultry and human beings. Thus, permanent monitoring of microbial air quality in chicken houses is necessary. [  The richness of biological genera in the urban atmospheric environment was lower than in concentrated poultry feeding operations. The bacterial lineages of bioaerosols present in the seven size stages for layers clustered apart from those for broilers, suggesting that the type of poultry house is a more important factor than the particle size in shaping the microbial communities. Results suggest that bioaerosols containing antibiotic resistance genes and potential airborne pathogens from animal feeding operations can be efficiently transferred to the nearby environment.
[54] The level of PM10 in poultry facilities did not exceed 4.5 mg/m 3 . After the flock entered the clean house, the level of endotoxins and β-glucans increased from below detection limit to 8364 ng/m 3 and from 0.8 ng/m 3 to 6886 ng/m 3 , respectively. The results show that professional activities in poultry farms are associated with constant exposure to bioaerosol, which may pose a health hazard to workers. In addition, it was found that workers' exposure to airborne microorganisms increased with consecutive stages of the chicken production cycle.
[31] Culture-based methods Molecular tools (qPCR) A significant positive correlation was found between litter fungal contamination (CFU/g) and air fungal contamination (CFU/m 3 ).
Spreading of poultry litter in agricultural fields is a potential public health concern, since keratinophilic (Scopulariopsis and Fusarium genera) as well as toxigenic fungi (Aspergillus, Fusarium, and Penicillium genera) were isolated. [38]

Culture-based methods
The lowest concentrations of total bacteria were obtained in those buildings where one-day-old chickens were kept. It was shown that for most of the investigated livestock premises the total bacterial concentrations exceeded the reference value of 1.0 × 10 5 cfu/m 3 . Furthermore, pathogenic microorganisms which are a potential threat to human health were found among the identified bacteria.

LAL
The endotoxin concentrations in the ambient air, and to which workers were exposed, appeared to be high in comparison with the threshold of 50 EU/m 3 over 8 h. Differences in dust and endotoxin concentrations between the cage and alternative systems may be due to the presence of litter and to the greater activity of the hens in the on-floor buildings. Effective methods to reduce worker exposure to air contaminants in laying houses still need to be developed.
[48] Concerning bacteria and fungi detected, 116 and 39 genera were identified, respectively. Among bacteria, Staphylococcus cohnii was present in the highest proportion (23%). The total inhalable bacteria concentration was estimated to be 7503 cells/m 3 . Among the fungi identified, Sagenomella sclerotialis was present in the highest proportion (37%). Aspergillus ochraceus and Penicillium janthinellum were also present in high proportions. Briefly, a limited amount of information exists on the bioaerosols present in a poultry production environment. Future work should include an expanded sampling plan and additional production sites for enhanced generalizability of the results. [66]

Culture-based methods
The highest median indoor concentration of culturable airborne bacteria (6.23 × 10 5 CFU/m 3 ) was found at the occupied poultry farm. Meanwhile, the highest median indoor concentration of culturable airborne fungi (3.15 × 10 4 CFU/m 3 ) was found at the flourmill site. In short, workers in Egyptian agriculture-related industries are exposed to aerosolized particulate matter and microbial concentrations. [73]
[24] The median concentration of airborne mesophilic bacteria was 1.7 × 10 6 CFU/m 3 in the processing area of the "moving rail," which is 8000 times higher than the background concentration of residential areas (approx. 210 CFU/m 3 ). Results evidence that poultry slaughterhouse employees are exposed to high concentrations of airborne microorganisms throughout the entire work time without using a respiratory protective device. Aspergillus, Cladosporium, and Penicillium represented most of the fungi (96% and 82% in the swine sheds for winter and summer, respectively, and 69% in the poultry sheds). Many microbial concentrations exceeded the Korean indoor bioaerosol guideline of 800 CFU/m 3 .
[58] Culture based-methods Thirty-one species attributed to thirteen fungal genera were isolated from the poultry house air. According to evidence, the majority of the identified fungal species found in industrial environments are characterized as allergenic and exposure to their spores may provoke adverse health effects in susceptible individuals.
[47] The highest total dust concentrations were found in poultry houses in Switzerland with median concentrations of 7.01 mg/m 3 . The highest total and active fungus concentrations were detected in poultry houses compared to pig houses and greenhouses. Additionally, bacterial concentrations were high in all animal houses. The exposure level found in this study might put the farmers at risk of respiratory diseases.
[52] Results evidence that factors related to work in the housing areas of pigs and poultry (variables of ventilation and feeding management) were significantly associated with decrements in lung function.
Additionally, higher temperatures inside the pig houses were significantly negatively associated with lung function in pig farmers. Overall, lung function results were significantly associated with ventilation of the animal houses.
Regarding metabolite characterization, Limulus amebocyte assay was frequently used for endotoxin assessment (11 out of 58, 19%), while one study used ELISA assay for mycotoxin assessment [40]. On the other hand, chemical analysis of litter samples was performed by one study [62]. Furthermore, quantitative kinetic Glucatell assay was used for β-glucan assessment [31]. Additionally, some studies performed questionnaires [50,74] and spirometric measures from workers were performed [50,74] .

Discussion
Industrialization has led to increased animal density in enclosed production buildings, resulting in high concentrations of viable and non-viable bacteria and fungi, as well as metabolites in bioaerosols [21]. The poultry industry has been found to pose a significant global health risk due to microbiological contamination [73]. Farm facilities housing multiple animals promote complex mixtures of microorganisms in bioaerosols, including dust-containing feathers, skin fragments, feces, feed particles, microorganisms, and chemicals [74]. Long shifts in manufacturing plants have become common, resulting in workers inhaling complex bioaerosols, which can pose several health hazards in agricultural environments [21]. This situation has prompted increased studies on occupational health. Bioaerosols from farms can also pose health risks to nearby residents [53,74], highlighting the importance of research on human health, environmental impact, and the One Health approach to address these concerns. Broilers and laying hens are susceptible to bacterial and viral infections of the upper respiratory tract, as indicated by several studies [38,51,62,70]. The transmission of pathogens can occur through inhalation, close contact with infected animals, feces, litter, or contaminated objects, and inadequate biosecurity controls can result in significant economic losses [74]. As international trade expands, food safety concerns regarding the rapid spread of foodborne pathogens through the global food chain are increasing [73].
Moreover, environmental health concerns arise from the utilization of animal byproducts, such as poultry manure and litter, in agriculture. Repeated use of these byproducts as manure can lead to the accumulation of contaminants in agricultural soils, po-tentially increasing their bioavailability and toxicity in the environment [74]. Air sampling has been widely used to characterize occupational exposure to fungi, but it is important to consider the appropriate sampling period and the influence of variables such as ventilation and building features. Passive sampling methods, such as settled dust assessment, have been shown to be more reliable for collecting contamination over a longer period of time. Broiler manure and animal bedding have been identified as the primary sources of indoor air microbial contamination in the poultry industry [76][77][78][79][80][81][82][83].
It is recommended to use a multiapproach sampling protocol for a more comprehensive understanding of microbial contamination. While culture-based methods have been primarily used for microbial characterization, culture-independent methods such as cloning approaches and quantitative real-time PCR have shown to be suitable for various bioaerosol measurements. Molecular tools, such as whole-genome sequencing, could provide more information on the biodiversity of microorganisms in these environments. Overall, these findings highlight the importance of considering various sampling methods and assays in the assessment of indoor microbial contamination in the poultry industry [60,61]. Studies on bioaerosols in poultry production are limited and identifying all organisms, both viable and non-culturable, is important for characterizing bioaerosols in these facilities [60]. Inhalation exposure to non-viable microorganism components such as endotoxins and mycotoxins may cause health hazards, so evaluating non-viable components may be useful for assessing pulmonary disease risk. Microbial assessment of poultry farms shows the presence of numerous microbes, including zoonotic pathogens, which can act as transport agents of airborne diseases [49,61]. Despite the growing threat of fungal infections to human health, there are fewer studies conducted on fungi (and also viruses) compared to bacteria, and this lack of attention and resources makes it challenging to determine the precise burden of fungal infections and to encourage policy and programmatic action [75].
Several potentially pathogenic bacteria have been identified [24,30,75,77]. The potential dispersal pattern and distance of airborne bacteria and ARGs from these animal sources remain unknown [53]. However, it is important to note that clinically significant multidrug-resistant bacteria Staphylococcus sp. [53,75], E. coli [29], Campylobacter jejuni [71], among others, belonging to the WHO priority pathogens list of antibiotic-resistant bacteria (2017), were isolated from poultry farms.
Recently, the World Health Organization (WHO) published the first fungal priority pathogens list [79], listing 19 groups of human fungal pathogens associated with a high risk of mortality or morbidity. This formal recognition by the WHO highlights an important group of infections, which has been perennially neglected in terms of the awareness and research funding needed [80].
Concerning microbial components, endotoxin, a major component of the outer membrane of Gram-negative bacteria, poses a serious health risk [34]. Endotoxins found in airborne organic dust have been linked to respiratory disease in both humans and animals [34].
Regarding mycotoxins, some of the literature already evidenced occupational exposure in animal production facilities [40]. In fact, fungal species recognized as mycotoxin producers were reported in some of the selected studies [32,36,42,78]. Even though only one of the selected studies performed mycotoxin assessment, the obtained results are enough to hypothesize that workers in these settings may be at a higher risk of Aspergillus mycotoxicosis. Indeed, elevated concentrations of A. flavus and A. versicolor were recovered through environmental sampling. Additionally, through human biomonitoring, analysis of mycotoxins and/or their metabolites in blood and urine evidence detectable levels of the carcinogenic mycotoxin AFB1 [40].
Briefly, to mitigate and decrease such pollutants it is crucial to establish international standards for what constitutes good microbiological indicators from environmental samples, which could be used to guide risk reduction decisions and create effective incentives for people to follow such guidance, which have already been suggested [81].
Globally, temperature rises due to climate change have various impacts on ecosystems, human health, animal health, and food production, which also affect AMR [81].
The emergence of resistant fungal strains in occupational exposure scenarios has already been demonstrated [82,83]. Indeed, temperature increases may influence the susceptibility of pathogens (bacteria, fungi, and parasites) in chicken environments [84]. Thus, as in the case of bacteria, antifungal resistance should be addressed in further research [85,86]. Additionally, it is crucial to investigate the effects of heat stress on poultry production to formulate various effective mitigation strategies to reduce significant production losses [84].
The prevalent airborne microorganisms in animal production buildings are not well characterized in terms of quantity, composition, and risk group. Identification and quantification would be useful for determining the causative agents and performing risk assessments [27].
The poultry industry must be sustainable, and it needs to produce more with less, while benefiting all [87]. The sector must improve human, animal, and environmental health and welfare. Implementing a comprehensive and coordinated One Health approach that incorporates exposure assessment can help tackle threats to health and ecosystems [81], ensuring priority areas for action in order to mitigate microbial exposure, promoting a safe environment for workers and animals in poultry facilities, along with less environmental impact.
Overall, these findings highlight the need for improved biosecurity measures and environmental management practices to ensure animal health, food safety, and environmental sustainability in the poultry industry.

Conclusions
This review allowed us to identify microbiological contamination reported in the poultry industry, sampling methods and assays already employed to assess occupational exposure to microbial contamination within different scopes (occupational health, food safety, and animal health), and knowledge gaps to be tackled in future studies.
Poultry workers are exposed to several microbial contaminants in their workplace. Exposure to bacteria and fungi has been assessed and reported, as well as bacterial metabolites (namely endotoxins), β-glucans, and mycotoxins. Occupational exposure to microorganisms is a frequent concern, and the risk of exposure to potential pathogenic and resistant bioaerosols originating in poultry facilities is emphasized. Future research should aim to identify the main sources of contamination in this setting.
A One Health approach is a vital framework and the use of effective risk assessment tools and strategies can help prevent occupational exposure and protect the health of workers, consumers, and animals.