Figure 3. Spirochetes: A. Cross section of a spirochete showing the location of endoflagella between the inner membrane and outer sheath; B. Borrelia burgdorferi, the agent of Lyme disease; C. Treponema pallidum, the spirochete that causes syphilis

(Source: http: //www. bact. wisc. edu/niicrotextbook/disease/overview. html)

As it has been mentioned before, spirochetes are poorly stained with aniline dyes, thus either a special Giemsa-Romanovsky stain is used or native smears are investigated using in dark-field microscopy or phase-contract microscope.

Giemsa-Romanovsky stain is composed with methylene blue, eosin and azure and is performed as follows:

• Working solution of dye (2 drops of dye per 1 ml of distilled water) is placed onto smear for 10-20 min.

• Smear is washed with water and allowed to be air-dried.

Treponema is usually having pale pink color, Borrelia - violet and

Leptospira — pink.

2. Fungi: introduction, classification and morphology

Fungi are eukaryotic organisms that do not contain chlorophyll, but have cell walls, filamentous structures, and produce spores. These organisms grow as saprophytes and decompose dead organic matter. There are between 100, 000 to 200, 000 species depending on how they are classified. About 300 species are presently known to be pathogenic for man.

There are five kingdoms of living things. The fungi are in the Kingdom Fungi.

The taxonomy of the Kingdom Fungi is evolving and is controversial.

Formerly based on gross and light microscopic morphology, studies of ultra structure, biochemistry and molecular biology provide new evidence on which to base taxonomic positions. Medically important fungi are in four phyla:

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1. Ascomycota — Sexual reproduction in a sack called an ascus with the production of ascopspores.

2. Basidiomycota — Sexual reproduction in a sack called a basidium with the production of basidiospores.

3. Zygomycota — sexual reproduction by gametes and asexual reproduction with the formation of zygospores.

4. Mitosporic fungi (Fungi Imperfecti) - no recognizable form of sexual reproduction. Includes most pathogenic fungi.

Pathogenic fungi can exist as yeasts or as hyphae. A mass of hyphae is called mycelia. Yeasts are unicellular organisms and mycelia are multicellular filamentous structures, constituted by tubular cells with cell walls. The yeasts reproduce by budding. The mycelial forms branch and the pattern of branching is an aid to the morphological identification. If the mycelia do not have septa, they are called coenocytic (nonseptate). The terms "hypha" and "mycelium" are frequently used interchangeably.

Some fungi occur in both the yeast and mycelial forms. These are called dimorphic fungi.

The dimorphic fungi have two forms:

1. Yeast - (parasitic or pathogenic form). This is the form usually seen in tissue, in exudates, or if cultured in an incubator at +37°C.

2. Mycelium — (saprophytic form). The form observed in nature or when cultured at +25°C. Conversion to the yeast form appears to be essential for pathogenicity. Fungi are identified by several morphological or biochemical characteristics, including the appearance of their fruiting bodies. The asexual spores may be large (macroconidia, chlamydospores) or small (microconidia, blastospores, arthroconidia).

Most members of the kingdom Fungi lack flagella; the structures are completely absent in all stages of their life cycle. The only exception is the chytrids, which produce flagellated gametes. The absence of flagella then, is a synapomorphy which unites all the remaining groups of fungi. This has had a tremendous impact on fungal biology, because it means that no fungus can produce motile gametes, and two organisms must therefore come into direct physical contact to effect sexual reproduction.

There are four types of mycotic diseases:

1. Hypersensitivity - an allergic reaction to molds and spores.

2. Mycotoxicoses — poisoning of man and animals by feeds and food products contaminated by fungi which produce toxins from the grain substrate.

3. Mycetismus - the ingestion of toxin (mushroom poisoning).

4. Infection.

To summarize the following should be remembered:

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Rickettsia stained by Zdrodovsky method have red color; cytoplasm of cells where they are parasiting is blue; nuclei are dark blue.

Table 2. RESERVOIRS, VECTORS AND MAJOR DISEASES CAUSED BY RICKETTSIA

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3. Chlamydia. The family Chlamydiaceae consists of one genus Chlamydia with three species that cause human disease, C. trachomatis, which can cause urogenital infections, trachoma, conjunctivitis, pneumonia and lymphogranuloma venereum (LGV); C. pneumoniae, which can cause bronchitis, sinusitis, pneumonia and possibly atherosclerosis and C. psittaci, which can cause pneumonia (psittacosis).

Chlamydia are small obligate intracellular parasites and were once considered to be viruses. However, they contain DNA, RNA and ribosomes and make their own proteins and nucleic acids and are now considered to be true bacteria. They possess an inner and outer membrane like gram-negative bacteria and a lipopolysaccharide but do not have a peptidoglycan layer. Although they synthesize most of their metabolic intermediates they are unable to make their own ATP and thus are energy parasites.

Physiology and structure

1. Elementary bodies (EB) — EB are the small (0.3-0.4 μm) infectious form of the chlamydia. The possess a rigid outer membrane that is extensively cross- linked by disulfide bonds. Because of their rigid outer membrane the elementary bodies are resistant to harsh environmental conditions encountered when the chlamydia are outside of their eukaryotic host cells. The elementary bodies bind to receptors on host cells and initiate infection. Most chlamydia infect columnar epithelial cells but some can also infect macrophages.

2. Reticulate bodies (RB) — RB are the non-infectious intracellular from of the chlamydia. They are the metabolically active replicating form of the chlamydia. They possess a fragile membrane lacking the extensive disulfide bonds characteristic of the EB.

3. Developmental cycle — The EB bind to receptors on susceptible cells and are internalized by endocytosis and/or by phagocytosis. Within the host cell endosome the EB reorganize and become RB. The chlamydia inhibit the fusion of the endosome with the lysosomes and thus resist intracellular killing. The entire intracellular life cycle of the chlamydia occurs within the endosome. RB replicates by binary fission and reorganize into EB. The resulting inclusions may contain 100-500 progeny. Eventually the cells and inclusions lyse (C. psittaci) or the inclusion is extruded by reverse endocytosis (C. trachomatis and C. pneumoniae) (Fig. 4).

4. Protozoa have more complex functional organization on comparison with bacteria and fungi. Body of protozoa is covered by rigid membrane called pellicula, which is formed by outer layer of cytoplasm. Cell structure and organelles are identical to those of multi-cell animals.

Protozoa are included in the kingdom Protozoa, subkingdom Animalia, divided onto 7 types. Species pathogenic for humans (e.g. plasmodia, Cryptosporidia, trypanosoma, etc. ) are included in the 3 types: Apicomplexa, Sarcomastigophora and Ciliophora. For the time being, a total of 7,000 types are considered to be pathogenic for plants, animals and humans.

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Identification of protozoa is based onto morphological features of pathogens and depends on proper collection and fixation of clinical specimens.

Morphology of protozoa can be studied in their live condition or using variety of stains. Simple staining techniques (fuchsine or methylene blue) are not suitable, because it does not allow to detect a complex structure of these microorganisms. The simplest technique is staining by Giemsa-Romanovsky method. It should noticed that fixation of smears should be done not by flaming, but using special fixations mixture (e.g. ethyl alcohol with ether). In case of necessity to detect motility, the best results are achieved when sample stage is heated and microscopy is performed within 30 min. after specimen collection.

VI. Viruses.

Virus structure and replication are fundamentally different from those of cellular organisms (Table 3).

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