Pathogenic fungal diversity in excreta of free living wild birds in agro ecosystems of Ludhiana

Introduction

Birds play an essential role in agroecosystems, by provding a variety of functions, including predators, pollinators, scavengers, seed dispersers, pests controller, nutrient cycling, ecosystem engineers, and many others thus benefiting both the environment and agricultural productivity. The complex relationships between birds and microorganisms are increasingly becoming the subject of ecological research (Maul et al 2005) [1] The pathogenic and allergenic microbial species can act to reduce fitness, making individuals more susceptible to competition and predation, while severe infections/reactions are significant causes of mortality (Nuttall 1997) [2] . Fungi are important causes of diseases in wild birds and other species. The common birds such as pigeons harbor a number of fungal species which are of potential health hazard and poses a risk to humans (Joshi et al 2015) [3] . The various filamentous fungi such as Aspergillus sp. (31.1%), Mucor sp. (20.2%), Penicillium sp. (9.5%) and Fusarium sp. (6.7%) was isolated from pigeon excreta samples (Cermeno et al 2006) [4] . Similarly, Aspergilus, Rhizopus, Penicilium, Mucor and Fusarium species was isolated from Leptoptilos crumeniferus (marabou stork) droppings in Kenya (Nyakundi and Mwangi 2011) [5] . Aspergillosis is a complex of diseases which is caused by fungal pathogens such as Aspergillus fumigatus, A. flavus, A. terreus, and A. niger and most of the infections was caused by A. fumigates in humans and avian species (Melo et al 2020, Kanauji et al 2023) [6,7] . Recently, Aspergillus fumigatus has been listed as the third most critical pathogen by the World Health Organization (WHO) which plays a significant role in the prevalence of aspergillosis (Khan et al 2024) [8] .

The spores of Aspergillus when inhaled can cause serious and often fatal infections in a wide variety of captive and free-roaming wild birds (Arné et al 2021) [9] . Aspergillosis is a major cause of morbidity and mortality in birds (Tell 2005) [10] . A unique feature of avian aspergillosis is the presence of reproductive phases of Aspergillus sp. in tissues which is due to presence of cavernous air sacs, the warm core body temperature of birds (range= 38-45 °C) that provide a nutrient rich environment. The number of colonies for establishing a definitive diagnosis is usually between one and four; the recovery of large numbers carries a severe prognosis. Cryptococcus neoformans is opportunistic basidiomycete yeast that causes cryptococcosis, a life threatening infection. This yeast has 3 varieties known as C. neoformans var. grubii (serotype A), C. neoformans var. neoformans (serotype D), and C. neoformans var. gattii (serotype B and C). Cryptococcosis, caused by C. neoformans primarily affects the nervous system, leading to meningoencephalitis in humans and this infection can be fatal without proper treatment in some of the cases. An Inhalation or direct contact with spores of C. neoformans from bird droppings poses a major health risk to humans (Al-Temimay and Hasan 2016) [11] . The most important source of C. neoformans var. neoformans throughout the world is pigeon excreta and other avian droppings (Zarrin et al 2010 and Nawange et al 2011) [12,13] . Many bird species play an important factor in fecal contamination of drinking water sources and agricultural crops. Keeping this in view, it was planned to isolate and identify the pathogenic fungi (moulds and yeast) from the excreta of free living wild birds commonly found in agro ecosystems.

Materials and methods

Study area

Fresh excreta of different bird species (blue rock pigeon, Columba livia; rose ringed parakeet, Psittacula krameri; common myna, Acridotheres tristis; house crow, Corvus splendens; cattle egret, Bubulcus ibis and red wattled lapwing, Vanellus indicus) was collected from their roosting, foraging and nesting sites in agricultural crop fields and residential areas of Punjab Agricultural University (PAU) located in the main campus of PAU (30° 54' N latitude and 75° 48' E longitude) and dairy farm of Guru Angad Dev Veterinary and Animal Sciences University (GADVASU) (30° 90' N latitude and 75° 80' E longitude).

Collection of excreta of birds

A total of 15 fresh excreta samples consisting of pooled pellets of six different bird species were collected. The five excreta samples were collected from commensal bird species such as blue rock pigeon and house crow (n=5). The two excreta samples of cattle egret (n=2) and one excreta sample each of rose ringed parakeet, common myna and red wattled lapwing (n=1) were collected. The two dry excreta samples of blue rock pigeon (n=2) was also collected from the above mentioned sites of collection. After collecting the excreta samples in sterile plastic bags with the help of spatula these were labeled appropriately with the source, time and date of collection and then stored at 4 °C till further processing.

Isolation and characterization of fungi

Ten gram of each sample was thoroughly mixed in 90 ml of normal saline. Aliquots (1ml) from serial logarithmic dilutions of each suspension were pipetted onto the surface of duplicate Petriplates containing sabouraud dextrose agar (SDA) supplemented with chloramphenicol (50 µg/ ml) to suppress bacterial growth and Staib's media. The inoculum was spread evenly with a sterile T-shaped glass applicator on petriplates. The petriplates were incubated at 28 °C for 5 days to 3 weeks. The mean colony count of moulds was determined and represented as colony forming units (cfu/ g) of each sample.

Identification criteria of fungi

The moulds isolated were identified by studying their colony and microscopic morphology as described by earlier published description (Cermeno et al 2006, Mishra 2010) [4,14] . For the preparation of slide cultures, the technique of McGinnis (1974) [15] was used. The presence of brown-pink coloured colonies (Cryptococcus neoformans) appearing on Staib's medium were noticed and the isolated colonies were further tested by negative staining with Indian ink to study the presence of capsule.

Results and Discussion Moulds

The isolated moulds in excreta samples of different bird species were identified to be Aspergillus fumigatus, A.niger, A. flavus, Alternaria sp. and Geotrichum sp. (Figure 1 and Figure 2). However, the excreta of different bird species varied in percent prevalence and population densities/ cfu/ g of these different moulds. Three samples out of 5 samples of blue rock pigeon yielded one or more moulds (Table 1). A. fumigatus, A. flavus and Alternaria sp. were found to be commonest species occurring in 60% of the samples; followed by A. niger (40%) and Geotrichum sp. (20%) (Figure 3). fumigatus was 8 × 10 6 cfu/ g followed by A. flavus (5 × 10 5 cfu/ g) and A. niger (6.5 × 10 4 cfu/ g) in one excreta sample of red wattled lapwing whereas Alternaria sp. and Geotrichum sp. could not be detected indicating that the excreta of red wattled lapwing carries only Aspergillus species and the population density of A. fumigatus was highest in excreta of this bird species. The prevalence of A. fumigatus was highest (86.7%) followed by A. niger as well as A. flavus (46.7%), Alternaria sp. (33.3%) and Geotrichum sp. (20%) in the total excreta samples studied (Figure 4). In the present studies, the excreta samples of all 6 species of birds contained pathogenic moulds. Aspergillus (especially A. fumigatus) was detected in all 6 species studied. House crow showed highest prevalence of A. fumigatus (100%) while the red wattled lapwing showed highest population density (8 × 10 6 cfu/ g) of A. fumigatus thus depicting the harmful exposure of their spores in environment which may affect the other birds as well as including humans. A. fumigatus has been determined as the most pathogenic among the causative agents of aspergillosis with widespread resistant spores present in the natural environment (Akan et al 2002) [16] . Aspergillosis is a respiratory tract infection caused by the genus Aspergillus of which A. fumigatus is the causative agent of infections in wild birds. Free-living wild birds become infected with A. fumigatus and other Aspergillus sp. causing aspergillosis which is a major cause of morbidity and mortality in birds. A wide variety of birds (waterfowl,gulls, crows, raven, raptors, songbirds, upland gamebirds blackbirds, cowbirds, grackles, herons and shorebirds) have died of aspergillosis and probably all birds are susceptible to it. Young birds appear to be much more susceptible than adults (Friend 2001) [17] . Soltani et al (2013) [18] reported the occurrence of pathogenic fungi species such as Penicillium spp., Aspergillus spp., Mucor spp., Rhizopus spp., Alternaria spp., Fusarium spp., Cladosporium spp., and Paecilomyces spp. in the pigeon droppings collected from urban areas of Isfahan. Gómez-De-Anda et al (2023) [19] reported the occurrence of filamentous fungi genera such as Mucor spp., Rhizopus spp., Penicillium spp., Paecilomyces spp. and Scedosporium spp. with zoonotic potential in droppings of cattle egret collected from Mexico. The bird droppings have high concentrations of nitrogen compounds and nutrients due to which it provide fertile media for the growth and spread of various diseasecausing microorganisms including pathogenic fungi (Mirhosseini and Khosrav 2023) [20] . Moreover, the organic debris accumulated in bird nests creates a unique environment for development of various fungal groups (Błońska et al 2024) [21] .

Yeasts

The pink colonies of Cryptococcus neoformans showing presence of capsules with Indian ink staining were isolated in both wet and dry excreta of blue rock pigeon and in wet excreta of house crow but was not detected in wet excreta of rose ringed parakeet, common myna, cattle egret and red wattled lapwing (Table 3, Figure 5). Earlier studies showed isolation of C. neoformans in old and dry excreta especially that of pigeon (Zarrin et al 2010, Nawange et al 2011) [12,13] . Recently, it has been reported that C. neoformans showed a strong association with desiccated avian excreta (Chowdary et al 2012) [22] . The reason for the high occurrence of C. neoformans in avian excreta is believed to be due to a utilization of nitrogenous compounds such as uric acid and creatinine from bird excreta. Bird excreta could act as a potential carrier in the spread of pathogenic yeasts such as Cryptococcus neoformans into the environment which get transmitted to children and elderly as well as immunocompromised patients (Soltani et al 2013) [18] . Thus from the fungal analysis of excreta of different bird species, it can be concluded that excreta of birds in agroecosystems harbor a wide range of pathogenic moulds and infectious yeasts. As these birds are free living, so spread of infectious agents occur through faecal contamination of drinking water sources, pastures and agricultural crops thus enabling the direct transfer of infectious agents to take place which may be a source of infection to human beings as well as may be harmful to birds themselves.

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