With the exception of some parasites, particularly skin and intestinal parasites such as fleas and tapeworms (for example, taenia) which can be detected macroscopically, most disease-causing, infectious agents can only be identified by microscopy. In some cases it is sufficient to observe fresh organic material, such as faeces or, in the case of skin parasites, skin scrapings to detect and identify aetiological agents. However, cytology, meaning the observation of cellular structures following standardised sampling procedures, preparation and staining, is the method of identification of most aetiological agents of pathological processes in tissues.
This article describes the morphological features of the main categories of infectious agents; the aetiopathogenic role and morphology of skin and gastrointestinal parasites are dealt with elsewhere.
VIRUSES
Viruses, which are obligate intracellular organisms, can be formed of DNA or RNA and are located, respectively, in the nucleus and cytoplasm. They are small, being only a few microns in diameter, and do not have a cellular structure. They have a rounded shape and usually a uniform colour; given their appearance, they are often called “inclusion bodies”. Among the rare viral agents that can be recognized by cytology, the distemper virus, a Paramyxovirus (Fig.1), can be seen during the viraemic phase within circulating white blood cells or erythrocytes and are recognizable as a uniformly eosinophilic body: sometimes it is possible to identify similar viral inclusion bodies in the cytoplasm of the epithelial cells of some mucosae such as the conjunctiva.
It should be noted that viruses are rarely seen in cytological preparations: what is seen is the inflammation, with non-specific features, resulting from the infection. The numerous disease-causing viruses for which morphological information cannot be obtained by cytology, unless special techniques such as immunofluorescence or immunocytochemistry are used, include Coronavirus, which causes feline infectious peritonitis (FIP), Adenovirus, the causal agent of hepatic and respiratory infections, Herpesvirus, the cause of respiratory, conjunctival and cutaneous infections and Retroviruses, including feline immunodeficiency virus (FIV) and feline leukaemia virus (FeLV).
BACTERIA
Bacteria are the most common disease-causing agents that can be identified by cytological studies. They can also be found in normal conditions since they inhabit some anatomical districts, such as the oral cavity and anal area, as saprophytes or colonisers. Bacteria can have a spherical, rod-like, spiral or filamentous shape; they can be present as single organisms or in organized, structured aggregates, which facilitate their recognition. It is crucial to identify the bacteria when characterizing inflammatory processes, particularly if the bacteria have been phagocytosed by cells with this function: neutrophil granulocytes and macrophages.
The fundamental, morphological cornerstones of bacterial identification are the colour and the repeated shape and size of the organisms in the same context:
- colour: bacteria usually take on a basophilic hue which varies between species, but is identical within the same species;
- repeatability of shape and size: all the bacteria of a single species have the same size and shape; this is an essential criterion for distinguishing bacteria, especially spherical species, from precipitates of stain or cell debris.
Spherical bacteria (cocci): these are small, with a diameter of 0.5-1 micron, and intensely basophilic. They comprise the bacteria of the genus Staphylococcus, including the extremely common Staphylococcus pseudointermedius, which are usually present singly, but sometimes in pairs (diplococci) or aggregates of four (tetrads) (Fig. 2) and the genus Streptococcus, which have a tendency to form chains (Fig. 3).
Rod-shaped bacteria (bacilli): these are larger than cocci and have the shape of a short, straight rod (Fig. 4), 2-3 microns long, and a variably basophilic hue; this category of bacteria comprises most of the Gram-negative bacteria, including Escherichia spp., Pseudomonas spp. and Proteus spp. The rod-like bacteria include Mycobacterium spp., the aetiological agent of tuberculosis, which in the dog and cat can cause cutaneous and splanchnic infections: these bacteria, which are usually phagocytosed by macrophagic cells, are recognizable for having a rod-like shape but being colourless because of an external wall formed of a complex structure of peptidoglycans, glycolipids and mycolic acid, which prevents the uptake of the normal stains (Fig. 5). It is usually fairly easy to identify Mycobacterium spp., but in cases in which diagnostic confirmation is necessary or when there are few bacteria present, staining with Ziehl-Neelsen may be necessary for definite identification (Fig. 6).
Filamentous bacteria: these have a long, thread-like cell body (Fig. 7); this group of bacteria include the family Actinomycetaceae, such as Actinomyces spp. and Nocardia spp., which can have a segmented protoplasm (Fig. 8). These bacteria can sometimes cause septic, suppurative effusions as a result of migration of foreign bodies: samples of the effusion fluid are typically brownish and contain macroscopic yellowish granules, which are bulky colonies of bacteria.
Spiral-shaped bacteria: these have an elongated, wavy shape. One group of spiral-shaped bacteria are gastric Helicobacter-like organisms (GHLO) (Fig. 9), which have been given this name because they resemble the Helicobacter spp. responsible for a gastric disorder in humans. These bacteria can be found in samples of gastric mucosa from dogs and cats, but their real pathogenic role is still controversial
PROTOZOA
Protozoa have a characteristic cell morphology, with the structural features of the cytoplasm and nucleus often enabling morphological identification of the species. Some pathogenic protozoa commonly found in veterinary practice are described below.
Leishmania spp.; the amastigote has a small, ovoid cytoplasm and a hyperchromatic rod-like nucleus, with a small kinetoplast perpendicular to the nucleus (Fig. 10).
Toxoplasma spp.; these aetiological agents are elongated and tapered, resembling bananas. They have a weakly basophilic cytoplasm and a small round nucleus (Fig. 11). According to many people they are virtually indistinguishable from Neospora spp., and so, when a definitive identification is necessary, microbiological or molecular biology techniques are essential.
Giardia spp.: these protozoa are oval-shaped with a tapered lower extremity and two opposing nuclei (Fig. 12a); in optimal conditions of storage, preparation and staining, the long, filiform flagella can be detected. This aetiological agent lives adhered to the epithelium of the intestine. It can be detected during cytological examination of specimens of crushed mucosa taken with tissue forceps or, rarely, by examination of fresh stools; the identification is more difficult in this latter case (Fig. 12b).
Babesia spp.: these organisms have a pyriform cytoplasm with a small, condensed nucleus. During the phase that the parasites are present in the blood, they can be detected singly, in pairs or as tetrads within the cytoplasm of circulating red blood cells (Fig. 13).
Hepatozoon spp.: these organisms have a roughly oval, colourless cytoplasm in which an irregular nucleus can sometimes be detected (Fig. 14); during the parasitaemic phase this protozoon can be found phagocytosed by circulating monoctyes and neutrophil granulocytes.
FUNGI
The developmental and reproductive conditions of fungi give these organisms particular morphological features: from the point of view of cell morphology, they can appear as spores, rounded, quiescent forms that are present singly or in groups, or as hyphae, that is, chains (usually linear but sometimes branched) of cells divided by regular septae. Pathogenic fungi have a very similar morphology, which, with some exceptions, makes it difficult to identify the species or genus. The cell body of fungi, both in the spore form and in the hyphal form, can usually be identified by its basophilic protoplasm, surrounded by a cytoplasmic membrane and a colourless cell wall (Figs. 15 and 16). These morphological features are shared by many pathogenic species of fungi, such as Microsporum spp., Trichophyton spp. and Aspergillus spp.
Some groups of fungi are formed by a single cell, without an achromatic cell wall, which reproduces by budding; these include some “defective fungi” or “yeasts”, which include Malassetia spp. (Fig. 17) and Candida spp. (Fig. 18): the former are characterized by an oval cell body and unipolar budding which gives the organism an elongated, bilobed appearance; in contrast, the latter is spherical and displays unipolar, bipolar or tripolar budding which, only under certain condition, can evolve into the development of septate hyphae.
Mycoses caused by unicellular fungi, for example Histoplasma spp., Blastomyces spp., Coccidioides spp., and Sporothrix spp., are known in various geographical areas outside Europe. In Europe there are fungal diseases due to Cryptococcus spp., which is characterized by an irregularly basophilic protoplasm, a colourless cell wall and a bulky peripheral capsule (Fig.19), exploited by the fungus to defend itself from the phagocytic activity of inflammatory cells.
HELMINTHS
These aetiological agents are large, multicellular organisms (metazoa): cytological features of both the adult forms and the larval stages can be identified. Most of the parasitic helminths infest the gastrointestinal tract and they can be recognized microscopically by stool flotation studies during which the egg packets or sometimes the larvae can be identified, although the details of this go beyond the scope of this article.
It is difficult to classify the helminths according to cytological criteria, because they can be present in different forms and sizes depending on the stage in their life cycle. They are usually large, in the order of hundreds of microns, and have an elongated shape; sometimes it is possible to see the individual cells making up the body and sometimes they have particular anatomical features or conformations facilitating their recognition. The identifying morphological characteristics of some of the most frequently found helminths are described below.
Dirofilaria spp. The most commonly encountered pathogens of the genus Dirofilaria are Dirofilaria immitis, which, as an adult, localises within the heart chambers, and Dirofilaria repens, which infests subcutaneous tissue. Both produce larvae that circulate in the peripheral blood and that can be detected on a blood smear; they have an elongated body and, using enrichment techniques such as Knott’s test, can be distinguished on the basis of the morphology of their caudal extremities: D. immitis has a straight caudal extremity (Fig. 20), whereas D. repens has a hook-shaped extremity.
Aelurostrongylus spp. A. abstrusus is a pulmonary parasite of cats, which lives inside the respiratory tract; it produces eggs and larvae that are morphologically characteristic because of their “S”-shaped caudal extremity. The larvae ascend the respiratory tract and, through the pharynx and oesophagus, reach the bowel before being excreted with the faeces. As a result, A. abstrusus can be detected both in bronchoalveolar lavage fluid, where it is present rolled up on itself (Fig. 21), and, much more simply and with less risk for the patient, in stool examined using a flotation method (Fig. 22).
Angiostrongylus spp. A. vasorum lives in the branches of the pulmonary artery; the eggs and larvae can be found in the alveoli and can, therefore, be identified in bronchoalveolar lavage or by stool flotation studies.
Capillaria spp. C. aerophila is a parasite of the airways and its large brownish eggs with polar opercula can be observed (Fig. 23). C. plica, on the other hand, infests the urinary tract, with a predisposition for the renal pelves and the bladder mucosa (Fig. 24).
Filaroides spp. F. hirti, which infests the airways, is characterized by a short, generally linear body (Fig. 25); the adult parasite lives in the bronchi and alveoli and can be detected in bronchial lavage fluid or in needle biopsies of nodular areas of lung parenchyma, which develop as a result of the inflammatory response to the parasite.
Suggested readings
- Raskin RE, Meyer DJ. Canine and Feline Cytology, a Color Atlas and Interpretation Guide. 2th edStLouis, Missouri, Saunders, 2010.
- Cowell RJ, Tyler RD, Meinkoth JH. Diagnostic Cytology and Hematology of the Dogand Cat. 2th ed. St Louis, Mo: Mosby, 1999
- Greene CE. Infectious Disease of the Dog and Cat. 3th ed. St Louis, Missouri, Saunders, 2006
- Masserdotti C. Batteri, miceti e protozoi al microscopio. Atti del 49° congresso nazionale SCIVAC; Approccio moderno alle patologie infettive (batteriche, micotiche, protozoarie): nel cane e nel gatto. Perugia, 29-31 ottobre 2004:77-85