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Generalities of Pathogenic Fungi

Generalities of Pathogenic Fungi and Main Mycological Techniques

Mycology

Mycology is the science that deals with the study of fungi.

The word mycology derives from the Greek mykes = mushroom and logos = discourse; etymologically it is the study of mushrooms.

Fungi are eukaryotic organisms (with differentiated cells, nuclear membrane and cytoplasmic organelles), spore-bearing, devoid of chlorophyll, heterotrophic, unicellular or filamentous, with cell walls made of chitin or other complex carbohydrates, that feed by absorption. They reproduce asexually by spores and sometimes sexually.

Fungi play a very important role in our lives. It can be said that not a day goes by without us being directly or indirectly benefited or harmed by them.

Fungi play an important role in the slow but constant changes taking place in our environment. They are responsible for the breakdown of organic matter, they cause most plant diseases and some diseases in animals and humans. They participate in the fermentation of bread, wine, beer, cocoa and cheeses. They are used in the manufacture of organic acids, drugs (ergometrine and cortisone), vitamins, and are responsible for the production of antibiotics (Penicillin and Griseofulvin).

They are both destructive and beneficial to agriculture: primarily by causing great plagues that destroy crops, and beneficial in that they decompose plant residues due to their ability to use cellulose, thus making plant nutrients available in the soil. Finally, one cannot overlook the culinary value of certain fungi such as the common mushroom and other edible species.

The vegetative apparatus of fungi is made up of hyphae, the totality of which forms a vegetative mycelium. This can be of two types: unicellular (formed by isolated cells that reproduce by budding (Fig 1), such as yeasts), or filamentous (with septate or partitioned hyphae (Fig 2) or non-partitioned or coenocytic hyphae (Fig 3)). There is an intermediate type, the pseudomycelium (Fig 4) formed by blastospores, as in Candida spp.

The reproductive mycelium (fruiting mycelium) forms spores of sexual or asexual origin (conidia). These reproductive structures differ from the somatic ones and are used for the classification of fungi; few fungi can be identified if these structures are not available.

The spore (spora = seed) is a minute unit that propagates without an embryo and gives rise to a new individual of the same species.

The filaments that make up the fungal soma grow apically.

Sexual reproduction is known as teleomorphic.

Asexual reproduction is also called anamorphic.

Classification of the Kingdom Fungi

Six divisions are recognised within this kingdom:

True fungi: EUMYCOTA, with cell wall:

  1. Division CHYTRIDIOMYCOTA: primitive cell chains attached to a substrate by means of rhizoids that end in a point. Zoospores have a single whip-like flagellum.
  2. Division OOMYCOTA: coenocytic mycelium. Zoospores with two flagella, one directed forward and the other backward. This division was for a long time considered a fungus, but recently by consensus a new kingdom was created, separating it from the Kingdom Fungi and placing it in the new kingdom, the Kingdom Chromista, which is much closer to algae than to fungi.
  3. Division ZYGOMYCOTA
  4. Division GLOMEROMYCOTA
  5. Division ASCOMYCOTA
  6. Division BASIDIOMYCOTA
  7. Division DEUTEROMYCOTA

Asexual Reproduction

Asexual reproduction is that by which individuals of the same species originate from a single cell or from a multicellular thallus that fragments, each fragment giving rise to a new individual. The forms of asexual reproduction can be divided as follows:

Asexual spores:

1. Unicellular:

Without conidiophores:

With conidiophore:

By morphology: Pyriform, Club-shaped, Spherical, Oval

By surface: Smooth, Rugose, Echinulate

2. Multicellular:

Conidiogenesis

Mycological Techniques

Sampling

Sample collection must be carried out under aseptic conditions. In treated patients, treatment should be withheld for 4–5 days. If ointments or powders have been applied, wash with physiological saline and repeat if the result is negative.

Samples can be:

  1. Scales: collected by scraping with the edge of a scalpel or a slide.
  2. Hairs: pulled with forceps from the edges of the lesion, especially those that appear broken or have a whitish appearance.
  3. Nails: scrape the nail from inside and also from the periungual groove.
  4. Urine and cerebrospinal fluid: prior sedimentation.
  5. Sputum: collected in a sterile jar, adding an equal quantity of 0.5% N-acetyl-L-cysteine solution and centrifuge.
  6. Exudates, faeces or blood: collected in sterile jars.
  7. Biopsies.

Direct Examination

  1. Lesions can be observed by fluorescence using a Wood’s lamp (3,600 Å). Use: in a dark room, at a distance of 10–12 cm, observe fluorescence.
  2. Scales, hairs and nails, gently heated on a slide with a clearing liquid (KOH, lactophenol).
  3. C.S.F.: India ink, latex test.
  4. Exudates, sputum, faeces, etc.: Giemsa, Gram, etc.

Cultures

Culture media usually contain salts, a carbohydrate source (various sugars or honey), an inorganic nitrogen source (ammonium salts or nitrates, etc.), organic nitrogen (protein substances, peptones or amino acids), vitamins and trace elements.

Antibiotics are usually added to inhibit bacterial growth (chloramphenicol 0.5% g/l, or gentamicin 0.05% g/L) but only when they do not act on the species to be isolated.

Inhibitors of contaminant fungal growth are also added, such as cycloheximide or actidione, at a concentration of 0.05% g/l.

On some occasions colouring substances are also added: phenol red, tetrazolium salts, etc.

The pH must be slightly acidic to inhibit bacterial growth; in media with antibiotics, the pH used is usually neutral.

Incubation temperature is normally between 25–30°C, although some cases require 37°C.

The media used are usually solid except in special cases; these include:

Usually seeded in a tube with actidione and another without actidione. If yeasts are suspected, media containing tetrazolium salts may be used. If actinomycetales are suspected, use Sabouraud dextrose without antibiotics.

Dermatophyte identification media:

  1. Sporulation Sabouraud or cornmeal agar: production of asexual spores.
  2. Potato-glucose agar: pigment production.
  3. Rice grains-water: selection of growth.
  4. Yeast extract plus hairs: hair perforation.
  5. Dextrose-urea agar: urease.

Yeast identification:

  1. Inactivated serum: filamentation or blastosis.
  2. Rice agar tween-80 (RAT): chlamydospores and pseudomycelium.
  3. Zymogram C: sugar fermentation.
  4. Auxanogram C: sugar assimilation.
  5. Api-aux and Pasteur-Yeast-System: complete identification of yeasts.

Observation and handling of cultures:

A small portion of colony is taken, including even the agar on which filamentous fungi have grown; if yeast-like, handling is identical to that for bacteria. A few drops of lactophenol or cotton blue are placed on the slide, a coverslip is applied, it is heated over a flame, and once the agar has melted the specimen is examined under the microscope.

Most Important Mycoses in Veterinary Medicine

Fungal infections are called mycoses. They are classified according to (1) as:

Superficial Mycoses or Dermatomycoses

Systemic Mycoses

Mycotoxicoses

Toxic diseases caused by poisonous metabolic products of moulds.