Direct Visualization of Fungal Burden in Filamentous Fungus-Infected Silkworms

Abstract

Invasive fungal infections (IFIs) are difficult to diagnose and to treat and, despite several available antifungal drugs, cause high mortality rates. In the past decades, the incidence of IFIs has continuously increased. More recently, SARS-CoV-2-associated lethal IFIs have been reported worldwide in critically ill patients. Combating IFIs requires a more profound understanding of fungal pathogenicity to facilitate the development of novel antifungal strategies. Animal models are indispensable for studying fungal infections and to develop new antifungals. However, using mammalian animal models faces various hurdles including ethical issues and high costs, which makes large-scale infection experiments extremely challenging. To overcome these limitations, we optimized an invertebrate model and introduced a simple calcofluor white (CW) staining protocol to macroscopically and microscopically monitor disease progression in silkworms (Bombyx mori) infected with the human pathogenic filamentous fungi Aspergillus fumigatus and Lichtheimia corymbifera. This advanced silkworm A. fumigatus infection model could validate knockout mutants with either attenuated, strongly attenuated or unchanged virulence. Finally, CW staining allowed us to efficiently visualize antifungal treatment outcomes in infected silkworms. Conclusively, we here present a powerful animal model combined with a straightforward staining protocol to expedite large-scale in vivo research of fungal pathogenicity and to investigate novel antifungal candidates.

Clinical and Mycological Characterization of Chronic and Recurrent Dermatophytes using Various Staining and Microscopic Methods

“…An exercise was conducted using 15 randomly selected isolates, and 4 more different staining/microscopy were conducted to rate these methods for future studies. 29,30,6 There are different stains available for the identification of dermatophytes, but only some stains are routinely used in the lab for the identification of dermatophytes. Lactophenol cotton blue stain could be one of the most frequently employed staining methods for physicians and microbiologists to be used for identification compared to the other staining techniques.…”

Alternative in-vivo models of mucormycosis

“…This offers an advantage in contrast to the G. mellonella model where infections are usually injected into the hemolymph without any organs specifically addressed ( Matsumoto and Sekimizu, 2019 ). Due to the convenient size, organ extraction is feasible, enabling the study of dissemination of spores from the hemolymph (or midgut) to various tissues types ( Yu et al., 2021 ). The silkworm has been established for studying bacterial infections as well as number of medical relevant fungal species like Candida glabrata , Crpytococus neoformans and Aspergillus fumigatus ( Matsumoto and Sekimizu, 2019 ).…”

“…Moreover, Yu et al. devised an easy-to-use protocol for visualizing diseases progression of filamentous fungal infection and evaluating treatment results with antifungal agents ( Yu et al., 2021 ). Post-infection with A. fumigatus and L. corymbifera , they dissected and stained larval organs with calcofluor white for the visualization of fungal growth and microscopic examination.…”

Time to Conquer Fungal Infectious Diseases: Employing Nanoparticles as Powerful and Versatile Antifungal Nanosystems against a Wide Variety of Fungal Species

“…The human fungal pathogens are responsible for a wide variety of infectious diseases, including those that affect the mucosa, the skin, and the invasive tissue [15][16][17]. Because of their rapid development, resistance to high temperatures (37 degrees Celsius), ability to exploit the host's nutrition to their advantage, ability to penetrate tissues, and ability to avoid the host immune system, pathogenic fungi are successful in infecting humans and causing illness [18,19]. The Cryptococcus, Mucor, Aspergillus, and Candida genus are some of the fungal species that are responsible for the majority of the fatal illnesses that are caused by fungi [20][21][22].…”