The science behind poultry aspergillosis

Introduction

Fungal infections are common across the globe in poultry but, less prevalent to bacterial and viral infections (Abd El-Ghany, 2021; Agbato et al, 2024) [2,5] and emerged as major respiratory disease owing to extensive use of antibiotics, corticosteroids and changed in farming systems (Biswas et al, 2010; Radwan et al, 2016; Mersha and Dawit, 2017) [31,159,124] . Aspergillosis holds a very special place in poultry medicine being the main mycosis affecting birds. Initial contamination of farms may occur through the use of a moldy litter or introduction of day-old chicks whose down has retained the conidia in hatchery facilities (Arne et al, 2011) [14] . This disease is a fatal airborne illness, widely distributed in chickens young or old including broiler breeders (Martin et al, 2007) [125] , turkeys, ducks, geese, pigeon, wild birds (Zeinab et al, 2024) [227] caused by toxins produced from fungus affecting the lower respiratory system (Fedde, 1998; Kunkle, 2003) [65,112] and nervous system. All ages of avian species are susceptible (Pazhanivel et al, 2018, Talbot et al, 2018; Hauck et al, 2020) [148,204,84] mainly as respiratory infection (Nururrozi et al, 2020) [139] . Chickens are particularly susceptible to aspergillosis because of their natural behavior of soil pecking and scratching which exposes to spores in the environment. Stress is the main predisposing factor (Saif et al, 2003; Copetti et al, 2004) [112,43] that results from mismanagement in commercial operations or backyard poultry (Jordan et al, 2002) [100] . Clinical manifestations depend on the infective conidia inhaled, pre-existing disease and the immune status of the host (Romina et al, 2019) [178] . Aspergillus fumigatus infection (Amer and Karim, 2019) suppress immunity against Newcastle disease in chickens. An immunosuppressive metabolite from Aspergillus fumigatus -a galactosaminogalactan predispose birds to fungal, bacterial and viral infections (Javed et al, 2005; Fontaine et al, 2011) [93,69] . Aspergillus spores are ubiquitous in the environment and grow anywhere in hot organic matters under humid environments. Dry litter conditions promote aerosolization of spores (Barbale, 2002; Flammer 2002) [21,68] .

Organic substrates like egg yolk, cardboard boxes, litter, old feed and feathers fulfil their nutrient requirements (Santos et al, 1996; Latge, 1999, 2001) [187,114,115] . Acute aspergillosis occurs on ingestion of large number of spores or through eggshell penetration and the chronic form in immunologically compromised birds. Hatchery provides the optimum environment for Aspergillus spores to thrive upon their entry through the eggs or incoming air. Contaminated eggs are the major source of spores that enter air handling units and ventilation system. The temperature and humidity in ventilation ducts are ideal for propagation of the molds especially when organic derbies have accumulated. Few species of aspergillus have been used as dietary supplementation in birds. Aspergillus Niger improved broiler growth and meat quality (Saleh et al, 2011) [182] . A. awamori have probiotic and antibiotics effect (Saleh et al, 2014) [183] and combination of A. awamori with lactic acid bacteria increased unsaturated fatty acid content and reduces saturated fatty acid in egg yolk (Saleh et al, 2017) [184] .

Etiological Agents

Brooder pneumonia in poultry is caused by Aspergillus. These organisms are common soil saprophytes which grow on organic matter under warm and humid environment including the damaged eggs (Atlaman, 1997; Lobna, 2014) [17,118] . The most common pathogens are Aspergillus fumigatus (Cortes et al, 2005; Souza and Degernes, 2005; Tell, 2005; Arne and Lee, 2019) [45, 200, 209, ] [132, 41, 11] . Other species involved in a single or mixed infections in poultry includes A. Niger, A. glaucus, A. nidulans (J. Barton et al, 1992; Joseph, 2000) [94,221] . Smaller sized spores of A. fumigatus are likely to be trapped completely in the nasal cavity, some able to reach trachea and lung, this is the most implicated reason associated with aspergillosis (Richard and Thurston, 1983) [175] . A new strain, Aspergillus sydowii (Romina et al, 2019) [178] with disease producing capability was isolated often as contaminants of papers, nuts, beans and straws. Mixed infections are possible (Perelman and Kuttin, 1992) [150] . Some influencing factors that contribute to aspergillosis are (Beernaert et al, 2010) [27] :

Anatomical and physiological characteristics of the bird's respiratory tract

Exposer to moldy organic matters like old feed, damp litter and compost piles favor Aspergillus growth. Mash feed can be contaminated with high number of molds (Akan et [9, 135, 197,] . The biodiversity of litter microflora depends on material choice, litter aging and handling techniques (Dennis and Gee, 1973) [53] . Inhalation of large number of spores over a short period of time or chronic exposure to low level of spores remain the principal risk factor of disease development (Barakat et al, 2012; Queiroz et al, 2013) [20,158] . If the spores are localized in the lungs, the fungi may be disseminated into other parts of the body, often leading to death (Powell et al,1994) [154] .

Poor ventilation: Stuffy air and ammonia promote spores build up in the barn. In aspergillosis free farms, the concentration of aspergillus species in air inside poultry house (Sauter et al, 1981 [189,52,75,138,136] varied from 10-10⁴ CFU per cubic meter. These fluctuations may be related to seasons and husbandry practices (Fulleringer et al, 2006) [73] . Concentration of aspergillus species in the air and litter decreases when windows were open (Pinello et al, 1977) [152] . Stressors like extreme cold, overcrowding, molting, poor nutrition, vitamin deficiencies (McMillan and Petrak, 1989) [127] , over use of antibiotics and corticosteroids (Oglesbee, 1997; Verstappen & Dorrestein, 2005) [142,222] and irritant disinfectants compromise the host immune system. Shi Bao et al (2014) [190] and Nemanja et al (2019) [134] reported immunosuppressive effect of dexamethasone by decreasing the chemotaxis and phagocytic activity of lymphocytes and macrophages. The inhaled spore may not cause disease, and remain dormant in the bird's air sacs and lungs until a stressful event triggers. Chickens that have undergone heat stress have reduced neutrophil activity, making them more susceptible. Poultry coinfected with Colibacillosis and Pasteurellosis more likely develop fungal disease.

Weather and season: Most of the fungal diseases occur sporadically with a seasonal variation (Mandal et al, 2004) [123] . Warm, damp and humid weather are ideal for prolific spore production (Vaezi et al, 2018) [219] . The global warming and high humidity aggravate various diseases (Kapetanov et al, 2015) [103] . The percentage of positive isolates in lung and environment samples was more in summer than winter season. Sultana et al (2014) [201] stated the presence of aspergillosis all through the year but highest in rainy season followed by summer least in winter. Thermal discomfort, starvation and migration (Young et al, 1998) [225] pamper the disease. Incidence of aspergillosis was more common in winter when indoor gas levels tend to be highest leading to bad ventilation, increased humidity and greater accumulation of organic matter in the litter (Zafra et al, 2008; Fang and Latge, 2018) [226,62] . Predominance of infection in closed housing during summer and presence of the fungi in the litter material during autumn make the eradication difficult (Solima et al, 2012) [199] . The distribution of the aspergillosis outbreaks is influenced by geographic location and seasonal variations, more frequent in areas with high temperature and rainfalls (Karwowska, 2005; Sajid et al, 2006) [104,181] . This seasonality may result from how climatic conditions and fungal ecology interplay with the bird's intrinsic factors (Chate and Bhivgade, 2010) [39] . Dusty litter or feed carrying spores transmit infection in poultry barns. In areas that are heated with brooders, in damp spots near drinking system, temperature and humidity rise (Ruediger et al, 2020), conidial proliferation becomes very high to infect young chicks (Poultry Affairs, 2022) [153] . Aspergillosis can also be produced by inhalation of spores from contaminated feed and poultry house litter (Thomas et al.). Large number of spores were found in wet litter and get aerosolized when the litter dries. Extremely dry air and dust, dry out respiratory mucosa causing the birds susceptible to the disease (Kristensen and Wathes, 2000; Munir et al, 2017) [109,131] . On parent stock level, wet litter or shavings are sources of aspergillosis. The eggshells become infected on litter and transfer this infection to hatchery. During incubation, the ventilation system becomes infected and on hatching, the dayold chicks will inhale the spore and develop pneumonia. Other sources in the hatchery includes wet carton. There is no true vertical transmission, but the eggshell can be the source of lateral transmission (Hybro, 2005) [89] .

Age:

The prevalence of aspergillosis in broiler is significantly influenced by age of the birds (Gull et al, 2024) [79] . Newly hatched chicks, turkey poults and ducklings are more susceptible. Higher body temperature in poultry allows quick fungal growth. Sultana et al (2014) [201] reported higher incidence of aspergillosis in 6-11 days of age and lowest in 0-5 days old chicks (Islam et al, 2003; Uddin et al, 2010) [90,215] . Increased susceptibility of young chicks to aspergillosis might be due to immature phagocytes or environment factors (Bennett, 1988) [26] . Chicken raised on saw dust than rice husk as bedding has increased incidence (Sultana S et al, 2014; Sajid et al, 2006) [201, 181] because of higher moisture content in saw dust. Wood shavings when stirred get dry out and spores become aerosolized and promote new cases (Rao et al, 1982; Dyar et al, 1984) [164,56] . Eggs in the incubator are common places for the fungus growth. The other chicks in the hatcher can be infected and equipment contaminated if the contaminated eggs burst out releasing high levels of spores (Bauck, 1994) [22] . Aspergillus is the most recovered fungal genus from dead in shell embryos (Abd. El-Aziz, 2015) [3] . Nutritional deficiencies (Ranck and Miles, 2001) [163] weaken immune system, and the malnourished chickens become susceptible to fungal infections. Lack of vitamin A and D, zinc, copper and selenium impair immune function in chickens. Lead poisoning is another factor (Samour, 2000) [186] . Feeding a balanced diet is crucial for maintaining optimum flock health and robust immunity (The poultry Pro, 2025) [211] . Certain infectious diseases such as infectious bronchitis, laryngotracheitis, Newcastle disease and fowl pox (Jacobson, 1980) [91] and subclinical infectious bursal disease aggravate aspergillosis (Okoye et al, 1991) [144] . Prevalence Among the diseases of poultry, aspergillosis constitute 4-7% (Talha et al, 2001; Badruzzaman et al, 2015) [205,19] . In broilers, prevalence of aspergillosis ranges from 4-8%, while 4% in layer chicks. The warm environment of summer and the highest indoor gas during winter (Girma et al, 2016) [77] favors the disease development. The occurrence and pathogenicity of A. fumigatus varied according to the lineage: in a challenged study, no clinical signs and mortality in layer chickens, more than 50% mortality occurred in broilers (Simon Thierry et al, 2013) [193] . Koenen et al (2002) [106] assessed the immunological differences between layer and broiler chickens. Layer chicken, showed two types of immune responsesantigen specific and cellular response against the only antigen specific immunity in broilers whose part of the immune response dysfunction due to genetic selection against body weight and feed conversion (Faublee, 1975) [64] . Birds are more susceptible to aspergillosis due to their anatomical and physiological makeup: no diaphragm, no epiglottis, poor vascularization, limited mucociliary function of the air sacs and heterophils replacing neutrophils (Tell et [209,44,210,128] .

Susceptibility to Chemical and Physical Agents

Some species of Aspergillus are quite resistant and can grow in sanitizing fluids (Rippon, 1982) [177] . Commercial disinfectants with Enilconazole have been used to control spores in poultry houses and surrounding (Redmann et al, 1989) [167] . Cinnamaldehyde, an essential oil from cinnamon inhibits Aspergillus growth (Mahmoud, 1994) [121] . Formalin fumigation is effective and cost efficient.

Pathogenesis

The fungus secretes enzymes like proteases and toxic secondary metabolites (Latge, 2001; Tekaia and Latge, 2005) [115, 208] . Clinical signs and torticollis detected in turkey poults without central nervous system lesions indicated toxin involvement (Richard et al, 1981, 1983) [170,175] . A. fumigatus produces (Rementeria et al, 2005) [169] gliotoxin an immunosuppressive cyclic peptide characterized by the presence of a disulfide bridge that regulate biological activity and toxicity (Gardiner et al, 2005) [74] . In poultry feedstuffs (Pena et al, 2010) [149] gliotoxin concentrations exceeding 20 µg/g, whereas 70 µg/g in tissues with air sac inflammation could be detected (Richard et al, 1996) [172] . Gliotoxin have cytocidal properties in chickens and turkeys causing mortality and torticollis after aerosol exposure (Richard, et al, 1996) [172] . Gliotoxin allow A. fumigatus to invade the epithelial cells of the lungs, and together with aflatoxin, suppresses the immune function of the host (J. Shankar, 2013) [97] . Galactosaminogalactan, another polysaccharide metabolite of Aspergillus fumigatus is also an immunosuppressive toxin (Fontaine et al, 2011) [69] . Extraction of gliotoxin from necrotic lesion of a cow udder infected with A. fumigatus suggest the biosynthesis and involvement of gliotoxin (Bauer et al, 1989; Beaver and Waring, 1994) [23,24] in inducing necrosis, but mechanism is not fully elucidated (Hurne et al, 2002) [88] . The increased intracellular calcium flux, mediated by gliotoxin has been linked. Gliotoxin interaction with thiol residue on the plasma membrane of the calcium channel cause oxidative damage and develop necrosis(Veronica et al, 2017) [223] . Aspergillosis is caused by inhalation of huge small, hydrophobic fungal spores into the respiratory tract (Oglesbee, 1977) that are trapped in nares, trachea and enter into primary bronchi and delivers to the posterior thoracic and abdominal air sacs which further to epithelial surfaces in the lungs (Nardoni. et al, 2006) [133] and there, germinate granulomas. These spores are engulfed by alveolar macrophages of respiratory tract and lung (Maina, 2002) [120] to find their way into the blood circulation and lymphatic stream, spread further into multiple organs (Dahlhausen et al, 2004) [50] like brain, pericardium, bone marrow, kidney and other soft tissues. A. fumigatus conidia resist killing by alveolar macrophages (Van et al, 1996) [218] . Tissue invasion creates an inflammatory reaction with heterophils, lymphocytes and monocytes to produce lesions (Kunkle and Rimler, 1996) [111] their severity depends on chronicity of infection, organs affected and the number of spores inhaled (Perez and Carrasco, 2000) [151] . The aspergilli migrate from lung to other parts of birds such as the liver, oviduct and ovary leading to disease syndromes characterized by acute to chronic infection, thereby suppressing growth and cessation of egg laying (Jordan et al, 2002) [100] . Besides inhalation and hematogenous spread to tissues, angio-invasive hyphae are also possible in disseminated mycosis (Dahlhausen et al, 2004) [50] . Aspergillus penetrate the eggshell (Amal et al, 2015; Brajen, 2025: Arrow) [10] , contaminate and infect the embryo (Hamet et al, 1991; Chauhan and Roy, 2016) [80,40] , eggs broken during incubation spread the disease in hatchery thus, the death of the embryos and chicks hatched with pathological lesions (Gray et al, 2021) [78] . In Ovo vaccination may present an increased risk of contamination. Two types of tissue reactions have been recognized: 1. Granulomatous or deep nodular form with neither exudative inflammation nor vascular lesions in the neighboring tissues. Reactions develop both in aerated and non-aerated organs like lungs and air sacs (Nardoni, 2006; Femenia et al, 2007) [133,66] . 2. Infiltrative or superficial diffuse formthe fungus frequently invades blood vessels and form aggregates of hyphae containing large numbers of conidia (Cacciuttolo et al, 2009) [36] that oftenimpending pulmonary blood flow leading to right ventricular dilatation and ascites. Another factor for mortality from aspergillosis includes exudates lodged in the trachea and syrinx producing respiratory embarrassment in birds (Pattisson et al, 2009) [147] . Clinical Signs Birds placed in environments contaminated with aerosolized conidia develop symptoms after a short exposure. Acute aspergillosis has 2-5 days of incubation period in chicken and turkeys (Richard et al, 1984; Melloul et al, 2014) [176,129] . Birds of different age groups are susceptible (Charlton et al, 2008) [38] involving many body systems. Manifestations of the disease depend on which organs involved and the infection either localized or disseminated. Extensive involvement of the respiratory tract occurs before symptoms are apparent. However, aspergillosis is generally reflected as a disease of respiratory tract (Singh and Malhotra, 1980; Sajid et al, 2006) [195,181] and occasionally of the central nervous system (Atlaman, 1997) [17] . Clinical signs depend on the infective dose and distribution of spores, presence of pre-existing diseases and the immune response of the host (Dahlhausen et al, 2004) [50] . The main clinical signs of the disease in broilers (Eassa et al, 2017) [57] include a third day onset of labored breathing and neurologic disorders as incoordination, torticollis and paralysis. Three forms of aspergillosis based on local involvement to systemic dissemination (Leishangthem et al, 2015; Fagbohun et al, 2020) [116,61] are as follows: Acute aspergillosis occurs by inhaling large number of spores. Within the first 3-5 days newly hatched chicks infected in the hatchery show very rapid and difficult breathing with gasping due to obstruction of the air passage (Aguilar and Redig, 1995) [6] . When these signs are complicated by other respiratory diseases -infectious bronchitis, infectious laryngotracheitis, there is gurgling or rattling noises with scratch sound during expiration (Okwara, 2016) [145] , but in aspergillosis dyspnea there is no respiratory noise (Vegad, 2015) [220] . Respiratory distress led to cyanosis (Richard, 1997) [173] . Young chicks and turkey poults that inhaled large number of spores during hatching or when placed on bedding contaminated with spores, potentially exhibit high morbidity and mortality often without showing clinical signs. Symptoms of anorexia represent 50%, dyspnea 48%, lethargy 40%, gasping 29% (In Video: Gasping and dyspnea) and torticollis 3% of signs in birds (Nururrozi, 2020) [139] . These findings are in consistent with Singh et al (2009) [196] , Beernaert et al (2010) and Melo et al (2020) [128] that clinical signs of acute aspergillosis manifest in a few days to 2 weeks of age. Nasal discharge occurs in acute aspergillosis (Sajid et al, 2006; Arne et al, 2011) [14,181] . Nasal aspergillosis causes exudative rhinitis and nasal sinusitis possibly leading to malformation of the nostrils and beak (Bauck et al, 1994) [22] . Mycotic keratitis can cause blepharospasm, photophobia, periorbital swelling, turbid discharge, swollen eyelids, cloudy cornea and cheesy exudates within the conjunctival sac (Bechman et al, 1994; Hoppes et al, 2000) [87] . Otomycosis invasive infection due to aspergillus species have been reported by Henry et al (2000) [85] . Per acute form in chicks may bring sudden death.

Chronic form is associated with immunosuppression (Vanderheyden, 1993) [217] and common in older birds (Hassanain et al, 2018; Kannoju et al, 2021) [83,102] . Clinical signs include anorexia, increased thirst, diarrhea, pyrexia, lethargy, ruffled feathers, dehydration, stunting and gasping. Ocular changes include ophthalmitis, photophobia, periorbital swelling with cheesy exudates in the conjunctival sacs (Backman et al, 1994; Hoppes et al, 2000) [25,87] and granulomatous dermatitis. Cloudiness of eye and blindness are not rare. Chronic form is sporadic, has lower mortality (Shoukat et al, 2018) [192] due to gradual loss of lung function and ascites (Vegad, 2015) [220] . In breeders, Akan et al (2002) [9] reported aspergillosis as chronic infection with brain lesions confining to cerebellum or cerebellar meninges. Chronicity is the result of long-term malnutrition, pre-existing illness, over exposure to antibiotics, steroids and stress (Bauck L, 1994; Rahul et al, 2024) [22] . The fungus may invade skin or surgical wounds causing necrotic granulomatous dermatitis or systemic aspergillosis. CNS granuloma formation in the brain, abscesses in the cerebellum were also noticed (Pattison et al, 2009) [147] . Neurological signs include paralysis, ataxia, tremor, torticollis, lameness, lateral recumbency, seizures, convulsions (Dyar et al, 1984 [56,96,9,213,32] . Torticollis and loss of equilibrium are the significant signs in experimental and natural aspergillosis in poultry (Kunkle, 2003) [112] . In farms, mortality may increase suddenly, peak over a few days and return to the initial state in a biphasic manner. In chicks contaminated during hatching, aspergillosis is highly fatal in the first 10 days of life causing 50-80% mortality (Kuldeep et al, 2013; Eassa, 2017; Wondimu et al, 2019) [57,224] within 5-14 days of symptoms development. Unilateral drooping of the wing due to infection of the thoracic and clavicular air sac or proximal humerus bone and repeated vomiting because of lesions in the anterior air sac also often accompanied (Forbes, 1991, 1992) [70,71] . Gross Lesions The primary location of lesions are the lungs, air sacs and other organsesophagus, proventriculus, gizzard, small intestine, liver, kidney, spleen, skin, trachea and peritoneum (Ahamad et al, 2018) [7] . Extensive involvement of the respiratory tract can be seen prior to the development of clinical signs. Typical lesions consist of white to yellowish granulomas (Arrow) of millet seed to a pea size on the serosa and parenchyma of multiple organs (Reece et al, 1986; Perelman and Kuttin, 1992; Singh H et al, 1994; Ashok et al, 2014, Musa et al, 2014) [168,150,194,132] in broilers and matured broiler breeders. These granulomas contain the organisms which could be rarely penetrated by therapeutic agents (Krishnan, 2005) [108] . The colonization of spores in the lungs and air sacs promote proliferation of the organism, development of granulomatous lesions in individual organs and tissues through evocation of the host defense mechanism (Cacciuttolo e et al, 2009) [36] . Pulmonary lesions are characterized by multiple hard creams to yellow circumscribe plaques a few mm to centimeter in diameter throughout the lung surface and inside, scattered on ventral surface of sternum and air passages (Beytut, 2007; Singh et al, 2009; Ruediger et al, 2020) [29, 196,] . The lungs, liver and spleen from aspergillus infected geese and emu appeared darker red than normal pink appearance (McDougle and Vaught, 1968; Sunitha et al, 2010; Emin Karakurt, 2021) [126, 60] . Aspergillosis has been the primary cause of death within 2-7 weeks of age ostriches (Saravia-Gomez, 1978) [188] . Dyspnea and gasping due to hyphae growth led to necrosis and inflammation in the air sacs and lungs (Ahamad et al, 2018) [7] . Necrotic cells in the respiratory tract cause hypoxia and birds increased respiratory frequency in attempt to compensate body oxygen, while the presence of plaques in the respiratory tract inhibit oxygen exchange of the lung and disrupt oxygen circulation (Chu et al, 2017; Eassa et al, 2017) [42, 57] . Aspergillus Niger produce oxalic acid which combine with calcium ions form insoluble calcium oxalate (Pradeep and Uma, 2009) [156] on being deposited in the lungs -disrupt its functioning. Abscesses in the cerebellum or cerebrum of 3-5 mm could be seen as circumscribed white to greyish areas in broiler breeders, turkeys and layers (Kureljusic et al, 2012; Gino L, 2023) [113,] . Ocular changes, such as mycotic keratitis with plaques can also be observed (Rafael Lera). Ascites in chickens has been a frequent after effect of hypoxia from aspergillosis leading to pulmonary hypertension and heart failure (Julian, 1993; Zafra et al, 2008) [101,226] .

Diagnosis

Diagnosis based on clinical signs and postmortem lesions are incomplete, may be confused with other infections. Therefore, confirmation requires combination of evidence from history, clinical signs, postmortem, hematology, biochemistry, serology, radiographic changes, or endoscopy and fungal cultures (Jones and Orosz, 2000) [99] . Field cases are diagnosed through postmortem findings of white to yellowish caseous nodules in the lungs or air sacs (Olias et al, 2010, 2011) [140] . History of the birds reveal stressful events, environmental factors associated with immune suppression, drug therapy and chronic debilitating changes (Hofle, et al, 2001) [86] .

Definitive diagnosis is based on the isolation of Aspergillus species by culture and detection of the organism during histological examination (Charlton et al, 2008; Beernaert et al, 2010; Kim et al, 2011) [38,105] on Saburou's glucose agar or antibiotics, incubated at 37°C for 24 hours show characteristic conidial head and colony (Fowler, 1993; Aguilar and Redig, 1995; Dahlhausen et al, 2004) [72,6,50] . However, negative culture cannot confirm the absence of aspergillus infection (Redig, 1994) [34] . Species can be identified by colony morphology and microscopic characterization of spores (Latge, 1999) [114] . Other serological tests that confirm aspergillosis infections are: Enzyme-Linked immunosorbent assay (ELISA): Brown and Redig, 1994; Redig et al,1997; and Arca-Ruibal et al,2006 [34,166,13] . Immunohistochemistry: Carrasco et al, 1993; Jensen et al, 1997; Beytut et al, 2004; Beytut et al, 2007 [37,96,28,29] .

Galactomannan assay and Plasma protein electrophoresis: Cray et al, 2005, 2009a.

Lateral and ventrodorsally radiograph (Jones and Orosz, 2000) [99] , Endoscopy (Taylor, 1993; Oglesbee, 1997) [207,142] detect abdominal air sac infection. Recent molecular techniques like RT-PCR, Internal Transcribed Spacer (ITS) gene-based PCR for broiler breeders, dead in shell and hatched chicks (Radwan et al 2019) [160] , environment and hospital patients (Hasab et al, 2019) [82] are now available to detect and quantify Aspergillus burden in lungs (Melloul et al, 2014; Tartor & Hassan, 2017) [129, 206] . The 26S gene of Aspergillus fumigatus was identified by amplification of 401 bp fragment in PCR test.

Treatment

The economic loss from aspergillosis is huge as treatments are ineffective and challenging in poultry (John, 2002; Arne, 2011; Ameji et al, 2020) [98,14] because of: (a). Spontaneous recovery can occur if exposure to fungus is avoided, (b). Limited knowledge on the pharmacokinetics of antifungal agents in different bird species, (c). The granulomatous inflammations create barrier for the drugs reaching the targeted fungus, and (d). Presence of concurrent infections, immunosuppression and the late stage at which birds are presented make the treatment challenging (McMillan and Petrak, 1989; Flammer, 1993 and Orosz & Frazier, 1995) [127,67,146] . Effective treatment involves a comprehensive approach, including antifungal medication, symptomatic treatment and supportive therapy (Krautwald et al, 2015; Jenks et al, 2018) [107, 95,] . Best results achieved on combination of two antifungal drugs, therefore, a judicious consideration for less toxic alternative approach is advised (Tell et al, 2019) [210] . When the granulomatous lesion dried, topical treatment in conjunction with systemic therapy gave faster recovery. Since drug resistance has been increasingly reported in human medicine (Snelders et al, 2008) [198] , standardized susceptibility testing is also becoming mandatory in avian medicine. Itraconazole and posaconazole 10 mg/kg orally every 12 hours for 10 days is the drug of choice (Sultana et al, 2015, Bunskoek et al, 2017) [202,35] . Amphotericin B, 1.5 mg/kg intravenously once daily for 3 days (Hasan et al, 2011) [81] . Clotrimazole as nasal flush or nebulization 30 minutes. Ketoconazole, 30 mg/kg in conjunction with Itraconazole is given for 21 days. Voriconazole, 10 mg/kg 12 hourlies through oral route is more potent but absorption and bioavailability depends on gastric pH (Lestrade et al, 2016) [117] . Fluconazole 15 mg/kg 8-12 hours orally. Nystatin 50000 IU per kg once a week in drinking water significantly reduced chick mortality (Oladele et al, 2022) [143] . Enilconazole may be sprayed to decontaminate indoor volume. Spraying of thiabendazole (Fate et al, 1987) [63] , nystatin and copper sulphate decrease fungal contamination of litter materials. In outbreaks, a 1:2000 solution of copper sulfate in water prevents the spread or 600 gm Copper Sulfate per ton of feed for 5-7 days (Dyar et al, 1984) [56] . A fresh solution from 50 gm Copper Sulphate dissolve in 250 ml vinegar + 700 ml of water: 2 ml of this solution mixed with Griseofulvin @ 2 mg/liter water for 5 days reduced mortality). Tetracycline 200 mg a liter of water treat aspergillosis in chicks (Babras and Radhakrishnan, 1967) [18] . Chlorine bleach @ 5ppm in drinking water is effective (Janmaat and Morton, 2010) [92] . Anti-mycotoxigenic, antifungal and antioxidant compounds from wild stevia restrain the growth of Aspergillus, Penicillium and Fusarium (Cowan, 1999; Dikhoba et al, 2019) [46,54] . Essential oils inhibit growth of toxigenic fungi and their mycotoxins (da Cruz et al, 2013; Prakash et al, 2015; Powers et al, 2019) [49,157,155] . Broiler breeders experiencing chronic aspergillosis and broilers from these parents had symptoms of gasping, lameness, mortality from hatch, suboptimal growth among the survivors responded positively to essential oils Fytera Perform® at 150 gm + organic acids (Salacid GG®) 4 kg per ton feed after 10 days. Continued dietary inclusion eliminated recurrence and concurrent bacterial infections in parent flocks. Essential oils could successfully treat aspergillosis in poultry and decrease infectious agents in the environment (Ebani et al, 2018) [58] . Cinnamon oil has the highest synergy with organic acids and potentially effective natural method of controlling post-harvest aflatoxin contamination in poultry feed. Essential oils have antioxidant, antimicrobial, analgesic, antipyretic, antiulcer, anticonvulsant and anticarcinogenic properties (Uma et al, 2017) [216] thereby reducing pathogenic bacterial growth, improving gut health, weight gain and meat quality in broilers (Zhai et al, 2018; Ding et al, 2022) [228,55] . Several organic acids inhibit the growth of aspergillus strains and minimize mycotoxin production, are effective against Salmonella and E. coli infections (El-Saadony et al, 2022) [59] .

Prevention

Among the fungal diseases of poultry, Aspergillosis, Candidiasis and Mycotoxicosis have impact on poultry production (Mersha and Dawit, 2017) [124] . These pathogens mainly target the respiratory and nervous organs of poultry (Shiva Chandra et al, 2004) [191] . Prevention is the best way of control (Girma et al, 2016) [77] by ensuring adequate ventilation, cleaning and disinfection (Beckman et al, 1994) [25] , reducing exposure to the fungus and associated risk factors, and early intervention to mitigate the outbreaks. Keeping the hatchery free of aspergillus prevent outbreak in chicks. Hatching eggs are collected and stored to reduce sweating, exposure to spore-laden dust, hatching equipment and air ducts be clean and disinfected. Contaminated premise is fumigated. Trace and eliminate the source of spores usually from nest boxes, litter, cardboard boxes, wooden walls or ceilings. In the setters and hatchers, either formalin or enilconazole (Clinafarm®) may be fogged to decontaminate indoor volumes without adverse effect on embryo development (Hybro, 2005) [89] . Removal of dust from poultry houses, improving ventilation and airflow reduces humidity which decrease fungal growth by 75% (Redmann and Schildger, 1989) [167] . An 0.5 cubic meters per bird per minute of fresh air exchange ensure adequate ventilation (Poultry Pro,2025) [225] . Providing a balanced diet rich in essential amino acids, minerals, vitamins and immunomodulators boost their immunity. Probiotics as supplements promote gut health and supplementation of arginine in diets enhance immune function reducing the susceptibility to aspergillosis (Danish et al, 2023) [51] . Improving feed management practices is crucial for preventing this disease (Agbato et al, 2024) [5] . Aspergillus fumigatus is the most dominant fungal pathogen in poultry (Radwan et al, 2019) [160] causing great economic losses. Poultry workers with weak immune system must use face mask to avoid inhalation of infective spores. According to P. Arne and Lee (2019), reducing spore load by sanitation of hatchery and rearing premises, removal of moldy feed and bedding, and good air quality reduce fungal disease. Host innate immunity is essential for defending against the pathogens in early stage of infection. When treating other illness, duration of antibiotic usage and doses of corticosteroids must be weighed against mycotic infections (Oglesbee, 1997) [142] .

Conclusion

Aspergillosis continue to remain life threatening to immunocompromised poultry. An increased action to identify and isolate new bioactive compounds to treat the disease is imperative. Many plant extracts and their constituents have shown therapeutic alternatives, but still remain challenging. Interactions among different plant derived compounds should be evaluated to maximize the antifungal efficacy while minimizing toxicity. Mycotoxins are the leading cause of immunosuppression in birds, making them prone to bacterial and viral infections. Formulating strategies with focus on diagnosis, prevention and control of fungal pathogens will find a solution. Provide no environment for the fungal spore to thrive in farm and hatchery will overcome the disease.

Acknowledgement

AcknowledgementNot available

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