A variety of fresh and fixed tissues, including lung, liver, kidney, spleen, and mesenteric lymph nodes, from a 7-year-old, 300 lb. llama were submitted to the Texas A&M Veterinary Medical Diagnostic Laboratory (TVMDL) for histopathological examination, bacterial culture, and sensitivity testing. Routine bacterial cultures were attempted from the lymph node and lung samples on blood and tergitol agar plates. There was no bacterial growth from the lymph node on either plates, but the lung sample yielded growth of a moist, brownish-white colony on both plates. The colony on blood agar increased in size with extended incubation. Five days post incubation the colony was noted as having white crusts at the edge indicative of a fungal colony (Figure 1). The microscopic morphology of hyphae were those of a Coccidioides species having multinucleate, barrel-shaped arthroconidia along the length of the hyphae (Figure 2). Histologically there was marked effacement of lymph nodes and the lymphoid tissues with pyrogranulomas characterized by a mixture of macrophages, lymphoid cells and neutrophils intermixed with necrotic debris. There were large numbers of a spherical organism with a relatively thick outer wall (spherules), measuring 25-30 μmin diameter, and internal sporulation (Figures 3 &4).
Coccidioidomycosis is a fungal disease that affects humans and animals. It is primarily caused by two species, Coccidioides immitis or Coccidioides posadasii. Coccidioidomycosis is common in dogs, but has been reported in various species including cats, horses, and South American camelids like llamas and alpacas. The Coccidioides species are endemic to parts of the Western Hemisphere, including the southwestern United States, northern Mexico and scattered regions in Central and South America. In the United States, the southwestern states of Arizona, California, New Mexico, southern Nevada, Utah, and Texas are considered endemic regions. Both species of Coccidioides cause almost identical diseases, but they usually occur in different overlapping geographical regions of United States. Coccidioides immitis is found mostly in central and southern California, whereas Coccidioides posadasii is found mainly in southern Arizona, western Texas and southern New Mexico.
Coccidioides species are dimorphic fungi having two phases in the life cycle. The saprophytic phase occurs in the environment and artificial laboratory cultures and the parasitic (yeast phase) occurs in the host animal tissues. The saprophytic phase consists of mycelia with septate hyphae which are 2-4 μm in diameter and bear arthroconidia. When the hyphae degenerate, the arthroconidia are released in the environment and act as a source of infection, especially during soil disruption and storms. The arthroconidia enter into the body of the host, mainly by inhalation, and settle in the lungs. They transform into the yeast form that can be phagocytized by neutrophils and macrophages, within which they rapidly transition into immature spherules. The immature spherules are 10-20 μm diameter, and when mature are typically 20-200 μm in diameter and develop numerous 2-5 μm endospores. When mature spherules rupture, the released endospores either form new spherules in tissues or mycelia if they are released into a suitable environment. The mycelia later convert to arthroconidia and perpetuate the life cycle.
In mammals the lesions of coccidioidomycosis are generally limited to lungs, which may vary from focal to multifocal. Systemic lesions that result from haematogenous spread of the spherules from lungs to other organs may also occur, as was evident in the present case. In dogs, the most common site of dissemination appears to be the bone and soft tissues over the infected bone fistula.
The cultures of Coccidioides species represent a biohazard for laboratory personnel because of production of arthroconidia in the culture media where there is a risk of aerosolization. In the present case, fungal culture was not requested, although it was isolated, because this organism readily grows on blood agar media. Situations like this create a challenge for laboratory personnel working with Coccidioides if fungal etiology is not on the list of disease differentials. All of the specimen handling for this case was done in HEPA filtered biosafety cabinets. After histologic and microscopic confirmation, the cultures were destroyed via an autoclave. In suspected cases of respiratory conditions in South American camelids, such as llamas or alpacas, additional care and proper personal protective equipment should be worn by personnel while performing necropsies. This is due to camelid’s vulnerability to coccidioidomycosis.
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REFERENCES
Fernandez et al. (2018). Pathology of coccidioidomycosis in llamas and alpacas. Journal of Veterinary Diagnostic Investigation, 30(4), 560–564.
Nguyen C, et al. (2013). Recent advances in our understanding of the environmental, epidemiological, immunological, and clinical dimensions of coccidioidomycosis. Clin Microbiol Rev, 26:505–525.
Butkiewicz CD, Shubitz LF (2019). Coccidioidomycosis in alpacas in the southwestern United States, Transbound Emerg Dis., 66:807–812.