Chapter 3

Selected Infectious Agents

Evaluating cytologic samples often results in the observance of microorganisms, some of which may be primary etiologic agents, opportunistic secondary overgrowths, or part of the normal flora of the location sampled. In some cases, the challenge is not only the identification of the organisms but also the recognition of the significance or insignificance of the organism in the overall pathologic process. This chapter is intended to aid in recognizing selected organisms found during the cytologic evaluation of samples from dogs and cats. It is beyond the scope of this book and chapter to discuss all of the organisms noted in cats and dogs; however, this chapter is intended to serve as a reference for the more commonly encountered infectious agents in canine and feline cytopathology. Discussions of the pathologic changes and conditions that these organisms produce are found in the following relevant chapters. Sample collection, staining and submission of samples for culture were previously covered in Chapter 1.

Identification of Organisms

Size, shape, and staining characteristics are important in the cytologic identification of organisms. The remainder of this chapter contains brief descriptions of organisms, including routine staining characteristics, which can be identified by cytologic analysis, and one or more photomicrographs of each organism to aid in its identification. The staining characteristics indicated in this chapter are for commonly available, routine hematologic stains such as Wright, modified Wright, Wright-Giemsa, Diff-Quik, or Dip-Stat stains, unless otherwise stated.

Bacteria

Staining Characteristics

With the routine hematologic stains described previously, all bacteria, whether gram positive or gram negative, stain blue to purple, with a few exceptions such as Mycobacterium. The lipid cell wall of Mycobacterium spp. prevents uptake of hematologic stains, causing the organisms to appear as nonstaining “negative” rods.

Gram stains can be used, but it is much more difficult to find bacteria with Gram stains than with hematologic stains and Gram stains often do not give reproducible, accurate results for bacteria in exudates. Cells, exudative proteins, and bacteria (whether gram positive or gram negative) tend to stain red in cytologic smears made from exudates.

Primary and Opportunistic Secondary Infection versus Normal Microflora

Intracellular bacteria indicate an active bacterial infection (primary or secondary), whereas extracellular bacteria are nonspecific and may represent an active bacterial infection, normal microflora, or contamination. Concomitant intracellular and extracellular bacteria are indicative of true bacterial infection, whereas normal microflora and contamination yield only extracellular bacteria. Also, a monomorphic bacterial population (only one bacterial type present) suggests infection, whereas a pleomorphic population (mixture of rods and cocci or different-sized rods) may be seen with contamination, normal microflora, or a mixed bacterial infection. Mixed bacterial populations may occur with infectious conditions such as gastrointestinal (GI) infections, bite wounds, and foreign bodies.

Pathogenic bacterial cocci are usually gram positive and of the genera Staphylococcus, Streptococcus, Peptostreptococcus, or Peptococcus (Figure 3-1). Staphylococci usually occur in clusters of 4 to 12 bacteria; however, Streptococcus, Peptostreptococcus, and Peptococcus spp. tend to occur in short or long chains of organisms. Staphylococcus and Streptococcus spp. are aerobic, and Peptostreptococcus and Peptococcus spp. are anaerobic. When cocci are identified in cytologic preparations, aerobic and anaerobic cultures and sensitivity testing should be performed to identify the organism and to guide optimal antibiotic therapy. As most cocci are gram positive, antibiotic therapy effective against gram-positive organisms should be used when it is deemed necessary to start therapy before culture and sensitivity results are received.

Figure 3-1 A, Neutrophilic inflammation with moderate numbers of bacterial cocci present both intracellularly and extracellularly. (Wright stain.) B, Higher magnification showing a neutrophil containing phagocytized bacterial cocci. (Wright stain.) C, Scattered red blood cells, two neutrophils, and a superficial squamous epithelial cell are shown. One of the neutrophils contains phagocytized bacterial cocci. (Wright stain.) D, Bacteria cocci chains. (Wright stain).

Dermatophilus congolensis replicates by transverse and longitudinal division, producing long chains of coccoid bacterial doublets that resemble small, blue railroad tracks (Figure 3-2). It infects the superficial epidermis, causing crusty lesions. Cytologic preparations from the undersurface of scabs from these crusty lesions are most rewarding in demonstrating organisms. The preparations usually contain mature epithelial cells, keratin bars, debris, and organisms. A few neutrophils may also be found.

Figure 3-2 A-D, Impression smear from skin lesions showing bacterial cocci replicating by transverse and longitudinal division, producing long chains of coccoid bacterial doublets that resemble small, blue railroad tracks. This pattern of bacterial cocci is typical of Dermatophilus congolensis. (Wright stain.).

Small Bacterial Rods

Most small bacterial rods are gram negative and some can be recognized as bipolar rods (Figure 3-3). It is safe to assume that all pathogenic, bipolar bacterial rods are gram negative. Common small bacterial rods include Escherichia coli and Pasteurella spp. Infections with bacterial rods are usually associated with a marked neutrophilic inflammatory response. When small bacterial rods are recognized in cytologic preparations, the lesion should be cultured to identify the organism, and sensitivity tests should be performed to determine the optimal antibiotic therapy. If it is necessary to institute antibiotic therapy before the culture and sensitivity results are received, the therapy employed should be effective against gram-negative organisms as most pathogenic small rods are gram negative.

Figure 3-3 A, Bipolar rods in cat (Wright stain). B, Higher magnification of bipolar rods (Wright stain).

Filamentous Rods

Pathogenic filamentous rods that cause cutaneous or subcutaneous lesions are typically Nocardia or Actinomyces spp. Other anaerobes such as Fusobacterium and Mycobacterium spp. may also be filamentous but are rarely so. Nocardia and Actinomyces spp. generally have a distinctive morphology in cytologic preparations stained with routine hematologic stains. They are characterized by long, slender (filamentous) strands that stain pale blue and have intermittent small pink or purple areas giving a “beaded” appearance to the organism (Figure 3-4). This morphology is characteristic of both Nocardia and Actinomyces spp. and the filamentous form of Fusobacterium spp. When these features are recognized cytologically, cultures should be performed specifically for Nocardia and Actinomyces spp. as well as for other anaerobes.

Figure 3-4 A, A long filamentous rod staining pale blue with intermittent small pink or purple dots (arrowheads) and smaller filamentous rods (arrows) are shown (Wright stain). B, Composite picture showing filamentous rods (arrows) typical of Actinomyces or Nocardia spp. (Wright stain.) C, Multiple filamentous rods and ruptured cells are shown. (Wright stain.)

Mycobacterium spp. does not stain with routine hematologic stains. As a result, negative staining rods (Figure 3-5) may be observed in the cytoplasm of macrophages, inflammatory giant cells, or both. When epithelioid macrophages, inflammatory giant cells, or both are encountered in cytologic preparations that do not contain any obvious organisms, a careful search for negative images of Mycobacterium spp. should be made. Mycobacterium spp. stain with acid-fast stains; therefore, when the character of the lesion suggests that Mycobacterium spp. be considered, and negative images are not identified, an acid-fast stain can be performed to aid in organism identification (see Figure 3-5, C). Cultures, and/or polymerase chain reaction (PCR) testing of tissue samples for Mycobacterium spp. may be helpful. Specific breeds of cats and dogs, including Siamese cats, Bassett dogs, and Miniature Schnauzer dogs, appear to be predisposed to Mycobacterium infections.

Figure 3-5 A, Negative images of Mycobacterium organisms in macrophage from cat liver aspirate (Wright stain). B, Fine-needle aspirate of a consolidated pulmonary mass. An alveolar macrophage, which contains nonstaining bacterial rods identified as clear streaks through the cell (arrow), is indicative of a Mycobacterium infection. (Wright stain.) C, Cat liver Mycobacterium (Wright stain). D, Cat liver Mycobacterium. (Acid-fast stain.)

Because these organisms are often refractory to common antibiotic therapy and reliable culture has special requirements, cytology is very useful in indicating to the practitioner that special cultures are needed. Multiple species of mycobacteria cause disease in dogs and cats and many of these species are zoonotic, which raises possible public health concerns. (See Mycobacterial infections. In: Greene CE, editor: Infectious diseases of the dog and cat, ed 4, St. Louis, MO, 2012, Saunders.)

Large Bacterial Rods

Large bacterial rods that are pathogenic and sometimes infect the cutaneous and subcutaneous tissues include Clostridia spp., and infrequently, Bacillus spp. When large bacterial rods are thought to be pathogenic, both aerobic and anaerobic cultures should be performed. Also, the smears should be inspected for large rods containing spores (Figure 3-6), as this may indicate Clostridium spp. infection.

Figure 3-6 A, Scattered inflammatory cells, red blood cells (RBCs), and large spore-forming bacterial rods typical of Clostridium spp. (Wright stain.) B, Higher-power view from same case as in A. Scattered RBCs and two large extracellular spore-forming bacterial rods are shown. (Wright stain.) C, Same case as in A. Multiple phagocytized spore-forming bacterial rods are shown. (Wright stain.) D, Clostridium organisms from mass on dog (Wright stain).

Occasionally, the large nonpathogenic bacterial rods of Simonsiella spp. are observed in cytologic specimens (e.g., contaminated tracheal washes). Simonsiella spp. are part of the normal oral flora of many domestic species and divide lengthwise, yielding parallel rows of bacteria that give the impression of a single, large bacterium with the characteristic “footprint” (Figure 3-7). Finding these organisms in cytologic samples from various locations indicates oral contamination; for example, if found in nasal flush samples, it may indicate a nasal–oral fistula, or if found in a lesion on a limb, it indicates licking and potentially could complicate the interpretation of an atypical mesenchymal population as neoplastic versus reactive.

Figure 3-7 A, Superficial squamous epithelial cell with adherent bacteria. Simonsiella organisms are large striated rodlike organisms. Other bacteria are present, adhered to the squamous cell and free in the background of the smear. (Wright stain.) B, Two superficial squamous epithelial cells with many Simonsiella organisms. (Wright stain.) C, One superficial squamous epithelial cell with many Simonsiella organisms and bacterial rods. (Wright stain.) D, Higher magnification, Simonsiella organisms (Wright stain).

Yeast, Dermatophytes, Hyphating Fungi, and Algae

Mycotic agents produce lesions that tend to be more granulomatous; that is, the inflammatory reaction has more macrophages compared with bacterial lesions. However, neutrophils may still be the predominant cell type, and eosinophils may be plentiful with certain hyphating fungi. Mycotic lesions often contain low numbers of lymphocytes, plasma cells, and fibroblasts, and smaller organisms that tend to be phagocytized by macrophages, but can be found phagocytized by granulocytic cells as well. Many factors, including the infectious agent, the animal demographics, the location of the lesion, the chronicity of the lesion, and the immune status of the animal, will influence the nature of the lesion in these infections. A comparison of the common fungi that form yeasts in tissues is presented in Table 3-1.

TABLE 3-1

Common Fungi That Form Yeasts in Tissue

Sporothrix schenckii

In many species, Sporothrix schenckii infection (sporotrichosis) can cause raised proliferative skin lesions that are often ulcerated. In cats, lesions tend to have many organisms, and cytologic evaluation readily leads to a diagnosis. In dogs, however, organisms are scant, and cytologic preparations must be carefully screened, as often only the inflammatory reaction is recognized. If no cause for a robust inflammatory reaction is identified, the lesion should be cultured, or a biopsy of the lesion should be submitted for histopathologic evaluation.

In cytologic preparations stained with hematologic stains, S. schenckii (Figure 3-8) are round to oval shaped or fusiform (cigar shaped), 3 to 9 micrometers (µm) long and 1 to 3 µm wide and stain pale to medium blue with a slightly eccentric pink or purple nucleus. Caution in interpreting is warranted, as occasionally these organisms can be confused with Histoplasma capsulatum if only a few yeasts are found and if they are not classically fusiform.