Bugs! Identification of common infectious agents by cytology (Proceedings)

Bacteria

Mycobacteria do not stain with routine cytologic stains and can be difficult to visualize. However, careful examination of the cells and background material reveals the presence of distinctive negatively stained thin rod present both intra- and extracellularly. The organisms can be confirmed by acid fast staining. Siamese cats appear to have increased susceptibility to mycobacteriosis. Mycobacteriosis often induces a granulomatous or pyogranulomatous inflammatory response rather than the suppurative response seen against most bacteria. Langhans' multinucleate giant cells and large epithelioid macrophages are often seen.

Actinomycosis/Nocardiosis infection is often seen as subcutaneous masses or intrathoracic lesions due to penetrating wounds. The thin organisms have a characteristic branching beaded filamentous appearance that often appear in large mats that resemble amorphous debris.

Clostridia can be identified as large gram positive rods (1 x 4µm) that often contain a clear, oval spore at one pole of the bacterial. Clostridium is associated with cellulites, penetrating wounds, and GI overgrowth.

Yersinia pestis is an uncommonly detected organism, the zoonotic potential of this organism makes identification of the organism crucial. Yersinia pestis is a gram-negative bacillus cytologically recognizable as bipolar coccobacilli present both intra- and extracellularly with large numbers of degenerate neutrophils. In cats, pneumonic plague occurs in about 10% of cases and can be seen with or without the classic bubonic presentation.

Fungal

Coccidioides immitis is a soil-borne dimorphic fungus, found mainly in the arid, acid-soil regions of the United States (Arizona and California) and in some regions of South America. In endemic areas, infected appears to be relatively common while development of clinical signs is relatively uncommon. It is primarily a respiratory pathogen and infection is by inhalation of spores. The incubation period for development of respiratory infection is 1-3 weeks after exposure. Disseminated disease occurs after primary lung infection, especially in dogs. Boxers and Doberman pinschers may be predisposed to disseminated disease. Until recently, cats were thought to be resistant to infection with Coccidioides, but both susceptibility to infection and development of clinical signs has been reported in endemic areas. In both dogs and cats with disseminated disease, lesions in long bones (especially the metaphyses area) and skin lesions are common. Coccidioidomycosis is induces a pyogranulomatous or granulomatous inflammation. Coccidioides immitis spherules (sporangium) are large organisms seen extracellularly. Spherules range in size from 10 to 100 µm in Romanowsky-stained preparations and contain a thick double-contoured wall with finely granular, blue-green protoplasm. Occasionally internal endospores of 2 to 5 µm may be seen. Organisms are scarce in cytologic preparations and multiple slides may need to be examined to find the organism. Due to the organism's large size, scanning is best done at low magnification (e.g., 10X). Mycelia may rarely be seen in tissue.

Blastomyces dermatitidis is a soil-borne, dimorphic fungus. The mycelial phase occurs in nature and the yeast form in vivo, although hyphal stages may be seen (albeit rarely). It probably has worldwide distribution, although the number of cases reported outside of North America is relatively small. A second strain has been isolated from cases in Africa and probably represents a distinct serotype with geographic diversity. The endemic area in the United States includes the middle western, southeastern and Appalachian states. Blastomyces can infect numerous tissues, but the lung is the most frequently involved organ in primary infection. Infection tends to be via inhalation of spores. Although direct puncture and skin wounds can lead to cutaneous lesions. most cutaneous lesions are derived from pulmonary infection. In dogs, infection more often occurs in young, large-breeds. Blastomyces is uncommon in cats, however when seen in cats the disease tends to be systemic rather than localized and Siamese appear to be over-represented in the literature. The incubation period for Blastomycosis is relatively long and variable (weeks to months). The extracellular yeast forms are dark blue, round, and 5 to 20 µm in diameter, with a thick biconcave wall having a granular internal structure. Broad-based budding may be seen. The organisms are likely found in aggregates of mucus and necrotic debris and induce granulomatous or pyogranulomatous inflammation.

Cryptococcus neoformans is the primary pathogenic species among the genus Cryptococcus however species have been identified to induce disease. C. neoformans a soil-borne, yeast-like fungus found worldwide. It multiplies to large numbers in the feces of pigeons and other birds where it can remain viable for months (note, it does not live in the intestinal tract of birds). Infection is via inhalation of spores. However both Cryptococcus neoformans and Cryptococcus gattii have been reported in the nasal passages of dogs and cats in the absence of local or systemic infection, and sub-clinical infection and/or asymptomatic carriers have been reported. Cryptococcosis is seen more frequently in cats than in dogs. The disease is manifested most commonly as nasal and frontal sinus infections, particularly in cats, that often extends to the meninges, brain and lungs. The characteristic tumor-like granulomatous lesions may also involve the skin and subcutis. Organisms are readily identified in swabs of nasal exudates or imprints/aspirates from nasal masses. New methylene blue and India ink can be used to demonstrate the negative staining capsule, however, care must be taken not to mistake air bubbles and fat droplets for organisms. Cryptococcus sp. are round to oval yeast that range in diameter from 8-40µ(including the capsule). The organism has a granular internal structure that stains eosinophilic to purple and is surrounded by a thick, non-staining, mucoid capsule. The capsule material can give the sample a mucinous texture. Occasionally, narrow based budding may be seen. Unencapsulated or rough forms are 4-8µm and are difficult to distinguish from H. capsulatum. Fungal culture and serology are useful in this case. The presence and type of inflammation ranges from the observation of minimal to no inflammatory cells amid a field full of organisms to pyogranulomatous inflammation. The degree and type of inflammation may be related to characteristics of the capsule. The polysaccharide capsule inhibits phagocytosis and the capsular polysaccharide have been associated with the induction of suppressor T-cells.

Histoplasma capsulatum is a oil-borne, dimorphic fungus with world-wide distribution that infects both cats and dogs. It is seen more frequent in certain geographic regions, e.g., in the United States, primarily in the northeast, central and south-central states. The primary route of infection is through the respiratory tract, but systemic spread occurs quickly in most cases. The incubation period for histoplasmosis is relatively short (12-16 days). Infection of the reticuloendothelial system results in the formation of tubercle-like granulomas in infected tissues. Pulmonary disease is common in affected cats. In addition to systemic histoplasmosis seen in both species, a self-limiting syndrome of pulmonary histoplasmosis is also seen in dogs. The small yeast-like organisms are round to oval and 1 to 4 µm in diameter with a purple nucleus and lightly basophilic protoplasm surrounded by a thin, clear halo. Organisms are seen within macrophages and neutrophils as well as extracellularly. A narrow-necked budding may be seen. Histoplasma induces a mixed to pyogranulomatous reaction.

Sporothrix schenckii is a dimorphic fungus found worldwide. It typically enters via cutaneous wounds of the limbs and induces suppurate nodules in the skin, subcutis, superficial lymphatics and lymph nodes. Dissemination to internal organs occurs occasionally, particularly in the dog. Localized lesions are sometimes seen on the face of cats. Sporothrix has also been isolated in the nasal cavity of cats with sporotrichosis and is more commonly detected in those with cutaneous lesions. The organisms are similar in size and morphology to Histoplasmosis. Round to oval to cigar-shaped 2 X 10-µm organisms with a thin, clear halo, slightly eccentric purple nucleus, lightly basophilic cytoplasm, and thin clear halo are observed both within macrophages and extracellularly. The presence of cigar-shaped organisms differentiates Sporotrichosis from Histoplasmosis. In cats, organisms are often abundant. However in dogs, organisms are rarely seen and identification may require searching over several slides.

Aspergillosis and penicilliosis can occur as focal or disseminated infections in dogs and cats. While the respiratory tract is a primary target of infection, both fungi are frequent contaminants of the respiratory tract, therefore diagnosis should be supported by a combination of culture, cytological or histological identification of the organism, and the presence of an inflammatory reaction. Both fungi are morphologically similar, necessitating culture for differentiation. German Shepherd dogs are frequently afflicted with systemic aspergillosis. While aspergillosis often appears as an opportunistic infection, there are reported cases without obvious predisposing factors. Infection with Aspergillus sp. can be associated with purulent, granulomatous, or pyogranulomatous inflammation. Cytologically, fungal hyphae are branching, septate, 5-7µ in width, with straight parallel walls and globose terminal ends. Hyphae can stain either intensely basophilic with a thin clear outer cell wall or appear as negatively staining images against a cellular background. Hyphae may be difficult to identify when found in low numbers or in dense mats admixed with mucus, inflammatory cells, and cellular debris. Occasionally, round to ovoid blue-green fungal spores may also be observed.

Rhinosporidium seeberi can infect the nasal cavity of dogs resulting in single to multiple polyps in which numerous small, miliary sporangia can be observed on the surface. Infection appears to be related to contact with water and/or trauma to the nasal mucous membranes. Cytologically, preparations contain variable numbers of magenta staining spores that range in diameter from 5 to 15µm with slightly refractile capsules and contain numerous, round, eosinophilic structures (spherules). In some cases, the spores stain deeply eosinophilic, preventing visualization of the internal structures. Sporangia are variably sized (30-300µ), well defined, globoid structures that undergo sporulation to contain numerous small, round endospores. Sporangia are not commonly observed in stained smears because they are only present in low numbers and the wall of the sporangia are slightly refractile and do not stain well. Three different maturation stages of endospores can be seen cytologically. Mature endospores predominate in cytologic preparations. These structures are 8-15 µm in diameter, with a thick, hyalinized cell wall and a pale, magenta to non-staining halo. Internal structure can be difficult to visualize in thick areas of the prep, but when endospores are spread out, numerous small spherical eosinophilic globular internal structures can be seen. Immature endospores are approximately 2-4 µm in diameter with lightly basophilic cytoplasm, a pink-purple nucleus encompassing 1/3 to 1/2 the endospore, and 1-2 smaller round magenta structures. Intermediate endospores are rarely seen spherical, granular, basophilic structures approximately 5-8 µm in diameter with eosinophilic to globular internal structures and a variably sized, clear halo. Rhinosporidiosis incites a mixed inflammatory response consisting of neutrophils, plasma cells, and lymphocytes as well as lesser numbers of . macrophages, mast cells, and eosinophils . Rosetting of inflammatory cells, particularly neutrophils, around the spores may be seen.

Phaeohyphomycosis is an opportunistic infection of pigmented (dematiaceous) fungi such as Alternia, Curvularia, and Bipolaris spp that commonly reside in soil or water. The morphology of the fungi varies with the specific organism and typically requires culture for final diagnosis. Hyphal structures are typically septate and may produce globose structures that can be confused with yeast forms.

Algae/Mold

Protothecosis is an unpigmented algae most often found in sewage contaminated water. Infection is seen more often in animals that are immunocompromised due to therapy or other diseases. In dogs, both a cutaneous and systemic forms of the disease have been recognized, while in cats the disease tends to remain cutaneous. In dogs, the organism is often detected in scrapings from the rectum or colon. The organism can be found extracellularly or occasionally within macrophages and ranges from 5-10 µm in diameter and have a round to oval shape. Internal septation results in the production of variable numbers (2-20) of basophilic endospores that contain granular internal material, a dark nuclei, and a clear halo. Prototheca induces a chronic pyogranulomatous inflammatory response.

Pythiosis is a water mold. Within the United States, the organism is found primarily within the southeastern states. Animals become infected by drinking or cutaneous exposure to contaminated water. GI exposure frequently leads to systemic dissemination while cutaneous exposure may result in localized dermal nodules and draining tracts. Cytologically, the organism is recognized by the presence of broad, poorly septate and branching hyphae. Pythiosis induces pyogranulomatous inflammation that often includes eosinophils.

Protozoa

Toxoplasma gondii infects virtually all mammals and is of importance to both human and veterinary health. T. gondii is a member of the protozoan phylum Apicomplexa, which includes the Plasmodium spp., Cryptosporidium and Eimeria spp. The life cycle of toxoplasmosis includes two phases. The intestinal phase occurs only in cats and produces oocysts. The extraintestinal phase occurs in all infected animals (including cats) and produces tachyzoites and then bradyzoites. Toxoplasmosis can be transmitted by ingestion of oocysts (in cat feces) or bradyzoites (in raw or undercooked meat). Tachyzoites of Toxoplasma gondii look similar to the tachyzoites of Neospora. Tachyzoites are 1 to 4 µm, crescent-shaped bodies with lightly basophilic cytoplasm and a central metachromatic nucleus. Organisms may be found intracellularly (primarily within macrophages) and extracellularly. Additional diagnostics are needed to differentiate Toxoplasma and Neospora.

Neospora caninum infection is most commonly detected in animals less than 1 year of age or immunocompromised animals and results in progressive, frequently fatal, ascending paralysis. The disease in older animals is diverse but characterized by systemic involvement, including marked pulmonary infiltration with associated pneumonia. On cytology, tachyzoites are indistinguishable from those of Toxoplasma gondii and are 1 to 5 µm by 5 to 7 µm, oval to crescent-shaped structures with a central metachromatic nucleus and lightly basophilic cytoplasm. They may be found intracellularly and extracellularly and induce a mixed inflammatory response.

Leishmania is a protozoa that is more commonly detected in dogs than cats. At least two species of Leishmania have been detected in animals in the United States. The amistigotes are most commonly seen on cytologic preparations and have a characteristic appearance. These are primarily intracellular organisms but may be seen extracellularly if the cell ruptures. Amistigotes are 1-5 µm in size, contain a magenta nucleus and a bar shaped kinetoplast placed perpendicularly to the nucleus. Skin, bone marrow, and lymph nodes are common sites of involvement.

Pneumocystis carinii is a protozoal organism that is most commonly reported in young dogs, primarily miniature dachshunds and Cavalier King Charles spaniels. Several immune defects have been identified that may predispose some dogs to with Pneumocystis carinii including hypogammaglobulinemia, decreased lymphocyte proliferation, and reduced numbers of B lymphocytes. Infection results in a diffuse interstitial pneumonia. The abundant foamy fluid present in the alveoli often contains trophozoite and cyst forms. Trophozoites are pleomorphic in morphology, ranging from 2-7 µm in length with pale blue cytoplasm and dark punctate nuclei. Trophozoites can be seen within macrophages or extracellularly. Cysts are less commonly seen and are extracellular, 5-10µ in diameter and contain 4-8 round, 1- to 2-µm basophilic bodies (sporozoites) arrange din a circumferential cluster.