Mycobiota recovered from the trachea and lungs of pigeons (Columba livia) captured in a grain mill

Respiratory diseases are commonly related to grain mills in association with fungi. Pigeons (Columba livia) are a potential source of pathogenic fungi and a public health concern, causing economic damages in urban areas. We aimed to determine the tracheal and pulmonary mycobiota of pigeons captured in a grain mill, identifying potentially pathogenic fungi. Captured pigeons were euthanized for the collection of tracheal secretion and lungs in saline suspensions for culture (100μL) in Sabouraud dextrose agar with chloramphenicol (48h/28°C). Yeasts were evaluated after Gram staining in microscopy and cultured in HicromeCandida agar, while lactophenol cotton blue solution was used in filamentous fungi/molds. Mycological identification keys were used for the genus determination. 207 fungal (9 genus) were isolated from the trachea and lungs of 21 pigeons captured: 199 molds (96.14%) and 8 yeast (3.86%). Aspergillus spp., Rhizopus spp., Penicillium spp. and Candida spp. were the most prevalent, mainly in lungs. We confirmed yeasts and molds from pigeons, increasing the potential risks for chronic or acute diseases after contact with several fungi from pigeons. A greater control of the presence of pigeons in the work environment is necessary.


Introduction
Pigeons (Columba livia) are cosmopolitan and a potential source of pathogenic fungi, causing economic damages in urban areas as a public health concern, due to environmental contamination by the deposition of their feathers, excrements and even fungi scattered in the respiratory process (Ferreira et al., 2016;Lee et al., 2017). Their granivorous habits affect in natura or industrialized grain-food industries from the processing to the final consumer, besides the importance as a vector of pathogens (Ferreira et al., 2016). Studies are scarce about the dust from activity in grain mill, which is the main responsible for respiratory clinical conditions in workers in these industries (Straumfors et al., 2021), as well as in the surrounding community (Theisinger;Smidt, 2017;Permatasari & Kolibu, 2020), being fungi important etiological agents involved in these processes.
The grain processing is related to several respiratory diseases caused mainly by mycotic agents, and the presence of pigeons increases the problem (Ferreira et al., 2016). Thus, the present study aimed to characterize the mycobiota of the respiratory system of pigeons captured in a grain mill in Alagoas to identify the coexisting fungi in their respiratory tract, focusing two different sites.

Material and Methods
Our study is descriptive observational (Patel;Patel, 2019), when pigeons were captured at the Moinho Motrisa S/A, (Maceió, Alagoas, Brazil) by a specialized company and submitted to anesthetic overdose euthanasia with intravenous thiopental sodium (DCB 086338/40 mg/kg-1 ) via brachial vein under aseptic conditions, performed in Histology and Embryology Laboratory (LHE/ICBS/UFAL) and approved by Animal Ethics Committee (n°58/2015).
Confirmed death, tracheal samples were obtained by introducing a sterile cervical brush into the trachea after an incision in the medial portion ( Figure 1A), adapted method to collect by Garcia et al. (2007) and Zampieri et al. (2015), and samples were immersed in sterile saline (1 ml) for vortex (1 min). Lungs were extracted after incisions in the dorsal portion ( Figure 1B), with the adoption of 2 methods in both lungs: (a) Lung fragments were inoculated directly on Sabouraud dextrose agar with chloramphenicol (ASDC) at 5 equidistant points; (b) Lung fragments were macerated with a sterile swab in a microtube with sterile saline (1 ml) for vortex (1 min). Right and left lobes were evaluated separately, and carcasses were incinerated following biosafety standards.  (Algrawal et al., 2014), while lactophenol cotton blue solution was used in filamentous fungi (40x-1600x) and mycological identification keys were used for the genus determination (Hoog et. al, 2000;Kidd et al., 2016).

Results and Discussion
Twenty-one pigeons were captured for collection in 2 anatomic sites, a total of 207 fungal isolates were obtained from the tracheal and pulmonary samples (  Probably, there is a greater fungi proliferation in the grain mill, an environment with suspension of spores and a higher rate of fungi inhalation (Permatasari & Kolibu, 2020), although all the captured pigeons in the dependencies of grain mill were clinically healthy.  (yeast-like) and Neurospora sp.
The immunological status of the birds, the habitat and methodology used for collecting biological samples can have changed the colonization profile in birds. We performed the collection with the introduction of a cervical brush in the trachea, without any contact with the oropharynx, different from the methodology that Garcia et al. (2007) and Zampieri et al. (2015) applied in birds from rehabilitation centers (RC), with the introduction of a swab into the oropharynx, which may increase the risk of contamination with yeasts from the oropharynx (Melville et al., 2004;Silva et al., 2019). Garcia et al. (2007) reported 42.59% of wild birds in RC with fungi isolated from the trachea, yeasts (24.53 %) and molds (12.5%), mainly C. albicans and C. parapsilosis. Birds in rehabilitation are more susceptible to stress, and weakness and/or drug therapy by corticosteroids and antibiotics facilitate the yeast proliferation (Silva et al., 2014). An increase was also observed by Zampieri et al. (2015) in seabirds (40% of Candida sp.), which are more due to the aquatic environment (Monaphati et al., 2020).
A study in birds of prey (Silva et al., 2019) confirmed a higher occurrence of yeasts isolated in oropharynx (93.3%) compared to trachea (6.7%), the majority Candida spp. (80%) isolated from Rupornis magnirostris (Roadside hawk/Gaviãocarijó) (73.7%). All birds of prey were clinically healthy and normally do not inhabit aquatic environments (Silva et al., 2019), which could explain the low number of yeasts isolated from the trachea, similar to our research.
High concentrations of airborne fungi were identified at the grain mill from Lithuania, where allergenic genus such as Penicillium, Cladosporium and Geotrichum prevailed (Straumfors et al., 2021). Thus, it is possible that fungal propagules have been inhaled by the birds, indicating, therefore, that they could also be inhaled by grain mill workers and cause acute respiratory symptoms through occupational exposure by the inhalation of large fungal inoculum (Theisinger & Smidt, 2017;Permatasari & Kolibu, 2020;Straumfors et al., 2021). All fungi isolated from the respiratory tract of pigeons are saprophytic and coexist on diverse organic matters, and the majority is considered as potentially pathogenic and involved in acute respiratory symptoms, increasing the possibility of interfering in the health of workers or the surrounding community (Senbeta & Gure, 2014;Pelliser et al., 2016;Theisinger & Smidt, 2017).

Conclusion
Our findings confirm that pigeons are frequently colonized by Aspergillus and Candida species. An exposure to their conidia may provoke adverse health effects such as allergic rhinitis or asthma by opportunistic pathogens involved also in systemic diseases in immunosuppressed individuals. Therefore, a better maintenance of the area is necessary, avoiding pigeon access and dispersion of fungi, as well as performing microbiological studies on air quality in grain mill dependencies to guarantee the health of the workers and surrounding community.