Outbreak due to Fusarium solani on a Brazilian ostrich farm Surto por Fusarium solani em uma fazenda brasileira de avestruzes Brote de Fusarium solani en una granja brasileña de avestruces

The aim of this study was to present and discuss the laboratory finding of Fusarium solani as opportunistic agent of invasive mycosis in chicks and eggs on an ostrich farm located in Cosmópolis (São Paulo, Brazil). Dyspnea and tachypnea were the main signs and symptoms presented by the chicks. Multiple whitish foci were found scattered over the chicks’ air sac membranes, lungs and livers and over the external membranes of the eggs. Fungal mycelia were observed in the air compartments of some of the eggs. An isolate purified from a lung culture was subsequently identified as Fusarium solani based on its morphological and molecular profiles. It was likely that the high humidity and temperature, as well as the matte paint that had been applied to the hatchery and birth room walls, provided conditions favoring fungal growth. Strict environmental sanitary measures are necessary to maintain microbial control. Uncommon fungi such as Fusarium solani must be considered as opportunistic pathogens in commercial ostrich culturing.


Introduction
The ostrich (Struthio camelus) is the largest living bird in existence. This rustic producer of meat, eggs, leather and feathers has achieved a privileged position in the Brazilian agribusiness market in a single decade (Almeida et al, 2005). The greatest difficulties in ostrich culture have been observed during the reproductive phase, which is the period from egg deposition to 90 days-old, during the unhatched and young birds are particularly susceptible to infectious diseases (Cooper et al, 2009).
The management practices and the health of the ostriches are the most important factors in controlling the spread of disease and achieving success in farming (Evans et al, 2004;Andreatti-Filho, 2000). Fungi may be responsible for serious cases of intoxication and infection that are frequently fatal for birds, especially the more predisposed individuals, such as chicks. The Aspergillus genre is the most common fungal pathogen identified in invasive avian mycosis. However, the new husbandry production methods have increasing the opportunistic infections caused by uncommon agents (Gulbahar et al, 2000;Gonçalves et al, 2012).
The aim of this paper was to discuss the identification of Fusarium solani as opportunistic agent of invasive mycosis in eight ostrich chicks and eggs at a Brazilian commercial ostrich farm located in Cosmópolis (São Paulo, Brazil) which were showing declining production due to recurrent fungal infections with fatal outcome of eggs and neonates. The cases were two newborn chicks and six eggs containing fetuses that were in the final stages of incubation, all from a commercial breeding facility located in Cosmópolis (Sao Paulo, Brazil). The present article characterizes a qualitative case study and it development is supported by the following scientific methodology proposed on Estrela (2018)

Case Reported
The two chicks hatched spontaneously but presented clinical signs of acute respiratory distress within hours of hatching. Intense dyspnea and tachypnea, followed by sudden death ( Figure 1A), were the main signs. To confirm fetal death in the eggs that exhibited delayed emergence, they were removed from the hatchery and opened. During the necropsies of the unhatched chicks, blood was observed in their coelomic cavities ( Figure 1B). In addition, numerous whitish spots were distributed throughout their coelomic cavities, on the air sac membranes, livers and lungs ( Figure 2). Whitish spots, similar to those observed in the chicks, were observed on the outer membrane of the eggs. A grayish powdery material occupied a large portion of the air chamber in one of the eggs. Tissue samples (air sac membrane, liver and lung) were collected from a chick and one egg. All of the samples were sent to the reference veterinary laboratory in the Campinas region, in São Paulo (Brazil).
The samples were seeded onto nutrient agar plates and incubated at 37°C for 24 hours for bacterial evaluation, and they were seeded onto Sabouraud dextrose agar plates and incubated at room temperature for five days for fungal evaluation. However, new fungal tests were requested by the veterinarians, so tissue samples were sent to where they were cultured on brain heart infusion (BHI) agar plates and incubated at 30ºC.  Research, Society and Development, v. 11, n. 2, e11411225499, 2022 (CC BY 4.0) | ISSN 2525-3409 | DOI: http://dx.doi.org/10.33448/rsd-v11i2.25499 was included at the end of the amplification. After amplification, the fragments were sequenced following the protocol provided with the BigDye reagent kit (Applied Biosystems TM Life Technologies TM , Foster City, CA, USA). The ITS regions were sequenced using the PCR primers. Consensus sequences were obtained using AutoAssembler (Applied Biosystems TM Life Technologies TM , Foster City, CA, USA) and SeqMan™ software (Lasergene TM , Madison, WI, USA). These sequences were subjected to a BLAST analysis (BLASTn) for species identification using two different databases: the National Center for Biotechnology Information (NCBI) (http://www.ncbi.nlm.nih.gov) and the Centraalbureau voor Schimmelcultures (CBS) (http://www.cbs.knaw.nl/Collection/BioloMICSSequences.aspx?file=all) databases.

Discussion
Fungi are ubiquitous microorganisms, and their proliferation is highly influenced by environment factors, as high temperature and humidity. The farm where the outbreak occurred had received an international certificate of good management practices (ISO-9001). However, the walls of the incubator and the hatchery facilities were coated with matte white paint, which most likely did not permit adequate hygiene and sanitary management, eventually predisposing these units to the colonization and proliferation of undesirable pathogens. Microbial agents can be introduced into aviculture incubators and hatcheries through contaminated eggs, insects and/or inappropriately dressed employees, posing a considerable risk of infection for the chicks (Katz et al, 1996;Jeffrey et al, 1994;Khosravi et al, 2008).
High concentrations of microbial agents, even those of low pathogenicity, constitute an important risk factor for infection in animals and humans. Colonization of the cloaca, for example, can facilitate the contamination of the shells of laid eggs and is associated with infectious processes in the yolk sac and embryonic death. Immunosuppressive factors, such as young age, stress and antibiotic administration, aggravate opportunistic infections. Candida albicans, Rhodotorula spp. and Cryptococcus spp. have been identified in the normal microbiota of the oropharynges and cloacae of ostriches (Cooper et al, 2009;Lábaque et al, 2003;Evans et al, 2004). While attempting to isolate filamentous fungi from the oropharynges and feces of ostriches obtained negative results; however, possible cases of pulmonary infection by these agents should be considered because their lower respiratory tracts are particularly affected by the inhalation of propagules present in the environment. No specific studies that provide information regarding the diversity of the fungal microbiota of the ostrich body surface are available (Almeida et al, 2005).
Opportunistic fungal infections can affect various segments of animal husbandry. Aspergillus fumigatus is the mycelial fungus that is most frequently associated with embryonic death and with respiratory symptoms in neonates in aviculture (Lima et al, 2001;Khosravi et al, 2008). The acute respiratory form, which mainly affects the lungs and air sacs, is the clinical form most frequently observed in birds with aspergillosis. In the case of chickens less than two-weeks old, which are more susceptible than older chicks to this acute form of pneumonia, the rates of morbidity and mortality are generally quite high (Melville et al, 2004;Oliveira et al, 2006;Khosravi et al, 2008). The presentation of a clinical pulmonary infection caused by A. fumigatus is very similar to that produced by other filamentous fungi, such as Paecilomyces spp., Acremonium spp., Trichoderma spp. and Scopulariopsis spp., which are common in the environment but are rarely identified as avian pathogens (Oliveira et al, 2006;Gonçalves et al, 2012). Reports of proventriculitis and ulcerative ventriculitis in ostriches have been linked to the existence of granulomatous pneumonia caused by Mucor spp. (Reissig et al, 2004;Orós et al, 2004). In this study, the clinical presentation of the affected ostriches was similar to that described for aspergillosis in birds in other countries (Richard, 1997); however, this investigation indicated that F. solani was the etiologic agent.
In one (16.6%) of the six unhatched eggs, a grayish powdery material occupied a large portion of the air chamber.
This contamination was most likely attributable to the presence of the fungus in high concentrations in the facilities. The material, denominated a fungal mat and consisting of an accumulation of fungal structures, was located between the outer and inner layers of the air chamber. These findings are consistent with those observed by others in cases of infection by non-Fusarium mycelial fungi (Gonçalves et al, 2012). Fusarium solani has been described as an infectious agent in reptiles, fish and mammals. Severe multifocal granulomatous pneumonia caused by F. solani was observed in a sea turtle (Lepidochelys kempi) specimen (Smith et al, 1989).
Fatal infections by F. solani were also diagnosed in shark pups (Sphyrna tibur) that were born in the National Aquarium in Baltimore (Uhart et al, 2006). In a case of meningoencephalitis in a German Shepherd dog, F. solani was the only agent identified (Yokota et al, 2004). Immaturity, advanced age, malnutrition, chronic underlying disease, stress or any other disorder that directly or indirectly affects the balance of an animal's immune system can facilitate infections by microorganisms that are uncommon disease agents, such as Fusarium spp. (Khosravi et al, 2008).
Companies involved in animal production, being concerned with productivity and potential economic losses, have adopted measures for the increasingly strict control of pathogens within aviculture incubators and hatcheries. It is important to monitor the environmental fungal load in facilities where the risk of infection is high. The signs and symptoms observed in this study were suggestive of an invasive mycotic process. The isolation of F. solani from the ostrich chicks and infected eggs is a strong indication that this organism was the etiologic agent. Fusarium solani fungus is commonly found in the environment and sometimes is identified in opportunistic invasive infections in humans, but is rarely observed in animal infections, although that trend may change in the near future (Gulbahar et al, 2000).
Because ostrich farming was only recently introduced into Brazil, little is known regarding the biological and clinical aspects of this species in this country. Knowledge of the normal microbiota of these birds and the environmental fungal load, as well as the molecular genotyping of clinical and environmental isolates will facilitate future investigations of the less frequently reported opportunistic fungi, such as Fusarium solani, to guide the adoption of adequate sanitary measures to prevent new outbreaks.

Final Considerations
Because ostrich farming was only recently introduced into Brazil, little is known regarding the biological and clinical aspects of this species in this country. Knowledge of the normal microbiota of these birds and the environmental fungal load, as well as the molecular genotyping of clinical and environmental isolates will facilitate future investigations of the less frequently reported opportunistic fungi, such as Fusarium solani, to guide the adoption of adequate sanitary measures to prevent new outbreaks.