History
A 3-year-old 26.63-kg (58.59-lb) spayed female Golden Retriever from southern Georgia was evaluated because of multiple large subcutaneous masses. The dog had whelped approximately 4 months previously, and an ovariohysterectomy had been performed approximately 1 month afterward. The owners reported that the dog was allowed to roam freely in a rural area, with access to ponds and marshy land.
Clinical and Gross Findings
On initial examination, the dog was bright and alert, with generalized lymphadenopathy and pyrexia (39.8°C [103.6°F]). A large (15-cm-diameter, 8-cm-deep) subcutaneous raised mass extended from the craniolateral aspect of the left shoulder (Figure 1). Another smaller subcutaneous mass with irregular borders was present on the medial surface of the right thigh. The left tarsal region was distended and edematous, and the caudal mammary glands were firm. Results of a CBC and serum biochemical analysis were unremarkable. Fine-needle aspiration of each of the 2 masses yielded purulent fluid that was submitted for cytologic analysis as well as for aerobic bacterial and fungal cultures. Cytologic evaluation of those fluid samples revealed large numbers of degenerate neutrophils. Enrofloxacin (8 mg/kg [3.6 mg/lb], PO, q 24 h), metronidazole (15 mg/kg [6.8 mg/lb], PO, q 12 h), and carprofen (4.4 mg/kg [2 mg/lb], PO, q 24 h) were administered for 7 days, during which time the dog developed lethargy and anorexia. A follow-up evaluation after 1 week indicated that the 2 masses had increased in size by approximately 50%.
Photograph of a mass on the craniolateral aspect of the left shoulder region of a 3-year-old spayed female Golden Retriever.
Citation: Journal of the American Veterinary Medical Association 241, 4; 10.2460/javma.241.4.447
Photograph of a mass on the craniolateral aspect of the left shoulder region of a 3-year-old spayed female Golden Retriever.
Citation: Journal of the American Veterinary Medical Association 241, 4; 10.2460/javma.241.4.447
Photograph of a mass on the craniolateral aspect of the left shoulder region of a 3-year-old spayed female Golden Retriever.
Citation: Journal of the American Veterinary Medical Association 241, 4; 10.2460/javma.241.4.447
The dog was hospitalized, and IV fluid therapy was administered, along with ketoconazole (10 mg/kg [4.5 mg/lb], PO, q 24 h) and enrofloxacin (8 mg/kg, IV, q 24 h). The dog was anesthetized by use of medetomidine hydrochloride (0.5 mg/kg [0.23 mg/lb], IV) and butorphanol tartrate (0.05 mg/kg [0.023 mg/lb], IV); the dog was intubated, then anesthesia was maintained with isoflurane gas at 2% for the duration of the procedure. A tissue biopsy specimen from the left shoulder mass was collected and submitted for histologic evaluation. In addition, a drain was placed in the left shoulder mass and was flushed daily with chlorhexidine solution. Tramadol hydrochloride (5 mg/kg [2.3 mg/lb], PO, q 8 h) was added to the pain control regimen. The dog was discharged from the hospital after 3 days. However, after 48 hours, the dog was hospitalized because of worsening clinical signs and died 24 hours later. Postmortem evaluation was declined by the owners.
Formulate differential diagnoses from the history, clinical findings, and Figure 1—then turn the page →
Histopathologic Findings
The tissue biopsy specimen obtained from the left shoulder mass was processed for histologic examination. Sections of skin and subcutaneous tissue had multifocal to coalescing areas of necrosis and inflammation surrounded by fibrovascular tissue that extended to the deep subcutaneous tissue, occasionally affecting the subjacent skeletal muscle. Areas of inflammation were characterized by infiltration of numerous multinucleated giant cells, epithelioid macrophages, and neutrophils; fewer lymphocytes and plasma cells; and rare eosinophils (Figure 2). Plasma cells and lymphocytes predominated at the periphery of the lesion. Numerous negative images of hyphae were evident in the areas of necrosis and fewer within the areas of inflammation and within multinucleated giant cells. Hyphae occasionally invaded the wall of blood vessels, and vasculitis, thrombosis, and hemorrhage were present. Hyphae had irregular bulging walls and very variable diameter (5 to 20 μm in diameter), with a few ballooning segments. Some hyphae were folded and had irregular branching and rare septation. Hyphae stained well with Gomori methenamine silver stain and stained weakly with periodic acid–Schiff stain.
Photomicrographs of sections of the shoulder mass (A–C) and lactophenol cotton blue stained smear (D) of the isolate obtained from the shoulder mass of the dog in Figure 1. A—In this tissue section, an extensive area of necrosis (top right) is surrounded by mixed inflammatory infiltrate with numerous neutrophils. Negative images of hyphae are visible within the area of necrosis. H&E stain; bar = 100 μm. B—The tissue in this section contains components of diffuse granulomatous inflammation, mainly multinucleated giant cells, macrophages, and plasma cells. Negative images of hyphae are visible within areas of inflammation (arrow) and within the cytoplasm of multinucleated giant cells (arrowheads). H&E stain; bar = 20 μm. C—Numerous intralesional hyphae with irregular bulging walls (5 to 20 μm in diameter) are present in this tissue section. Irregular branching and occasional rare septation are seen. Gomori methenamine silver stain; bar = 50 μm. D—Smear of the isolate obtained showing hyphae typical for Lagenidium spp. Lactophenol cotton blue stain; bar = 50 μm.
Citation: Journal of the American Veterinary Medical Association 241, 4; 10.2460/javma.241.4.447
Photomicrographs of sections of the shoulder mass (A–C) and lactophenol cotton blue stained smear (D) of the isolate obtained from the shoulder mass of the dog in Figure 1. A—In this tissue section, an extensive area of necrosis (top right) is surrounded by mixed inflammatory infiltrate with numerous neutrophils. Negative images of hyphae are visible within the area of necrosis. H&E stain; bar = 100 μm. B—The tissue in this section contains components of diffuse granulomatous inflammation, mainly multinucleated giant cells, macrophages, and plasma cells. Negative images of hyphae are visible within areas of inflammation (arrow) and within the cytoplasm of multinucleated giant cells (arrowheads). H&E stain; bar = 20 μm. C—Numerous intralesional hyphae with irregular bulging walls (5 to 20 μm in diameter) are present in this tissue section. Irregular branching and occasional rare septation are seen. Gomori methenamine silver stain; bar = 50 μm. D—Smear of the isolate obtained showing hyphae typical for Lagenidium spp. Lactophenol cotton blue stain; bar = 50 μm.
Citation: Journal of the American Veterinary Medical Association 241, 4; 10.2460/javma.241.4.447
Photomicrographs of sections of the shoulder mass (A–C) and lactophenol cotton blue stained smear (D) of the isolate obtained from the shoulder mass of the dog in Figure 1. A—In this tissue section, an extensive area of necrosis (top right) is surrounded by mixed inflammatory infiltrate with numerous neutrophils. Negative images of hyphae are visible within the area of necrosis. H&E stain; bar = 100 μm. B—The tissue in this section contains components of diffuse granulomatous inflammation, mainly multinucleated giant cells, macrophages, and plasma cells. Negative images of hyphae are visible within areas of inflammation (arrow) and within the cytoplasm of multinucleated giant cells (arrowheads). H&E stain; bar = 20 μm. C—Numerous intralesional hyphae with irregular bulging walls (5 to 20 μm in diameter) are present in this tissue section. Irregular branching and occasional rare septation are seen. Gomori methenamine silver stain; bar = 50 μm. D—Smear of the isolate obtained showing hyphae typical for Lagenidium spp. Lactophenol cotton blue stain; bar = 50 μm.
Citation: Journal of the American Veterinary Medical Association 241, 4; 10.2460/javma.241.4.447
Morphologic Findings
Heavy growth of a glabrous, colorless, moderately growing filamentous mold was obtained via both bacterial and fungal cultures of the purulent fluid samples collected from the left shoulder mass. Hyphae up to 45 μm in diameter were observed (Figure 2), but fruiting bodies were not produced in culture. A subculture of the isolate was submitted for further identification to the Fungus Testing Laboratory at the University of Texas Health Sciences Center and to the Pythium Laboratory at Louisiana State University. Amplification and sequencing of the internal transcribed spacer (ITS) region of the rRNA gene yielded sequence data with a high degree of similarity to Lagenidium giganteum. In addition, the ITS sequence was identical to that of isolates obtained from dogs with cutaneous infections caused by a novel Lagenidium pathogen.1 The isolate from the dog of this report was deposited into the Centraalbureau voor Schimmel cultures (accession No. CBS 127832).
Morphologic Diagnosis
Severe chronic multifocal to locally extensive necrotizing and pyogranulomatous panniculitis with intralesional fungal hyphae, consistent with a Lagenidium sp.
Comments
The genus Lagenidium belongs to the class Oomycetes in the kingdom Chromista, which also includes fish pathogens in the genus Saprolegnia, plant pathogens in the genera Pythium and Phytophthora, and the important mammalian pathogen Pythium insidiosum. This group of organisms is morphologically similar to but phylogentically distinct from the true fungi.2 The most well-studied species in the genus Lagenidium is L giganteum, which is a pathogen of mosquito larvae that has been applied environmentally as a biological pesticide.3 On the basis of similar morphology and an identical rRNA gene ITS sequence, the Lagenidium pathogen isolated from the dog of the present report appeared to be identical to isolates obtained previously from 6 other dogs.1 Although rRNA gene sequence data also indicated that this canine pathogen is closely related to L giganteum, differences in their in vitro growth characteristics (including cardinal temperatures) suggest that they are distinct. Unfortunately, the sexual structures needed to properly describe the full morphologic characteristics of this other canine pathogen have not been produced in culture; therefore, it has not yet been properly named or described. Future sequencing of additional genes and more detailed phylogenetic analyses may provide further information about the evolutionary relationship of this unnamed canine pathogen to L giganteum and to other oomycetes.
The epidemiological and clinicopathologic features of infection caused by this unnamed Lagenidium sp in dogs are similar to those associated with cutaneous pythiosis,1,4 including development of dermal or subcutaneous nodular lesions in young to middle-aged dogs living in the southeastern United States. Accessibility to water bodies could be a factor in acquiring the initial infection. Hematologic changes such as hyperproteinemia, neutrophilia, and eosinophilia can be observed in cases of lagenidiosis.1 No hematologic changes were detected in the dog of the present report. Lesions are often characterized by ulceration, edema, necrosis, and the presence of draining tracts and are generally invasive and progressive. Regional lymphadenopathy is a common finding. Unlike dogs with pythiosis, dogs infected with this unnamed Lagenidium pathogen typically have infection at noncutaneous sites such as great vessels, sublumbar and inguinal lymph nodes, lungs, pulmonary hilus, and cranial mediastinum.1 Gastrointestinal tract lesions have not been identified in dogs with lagenidiosis. Gastrointestinal pythiosis results in abdominal masses, and the main clinical signs are weight loss, vomiting, and diarrhea.
Cytologic examination of aspirate samples obtained from lesions associated with lagenidiosis in dogs typically reveals evidence of pyogranulomatous to eosinophilic inflammation. Hyphal elements with morphologic features suggestive of oomycetes (large diameter and infrequent septation) may also be seen. Histologically, Lagenidium infection resembles pythiosis and zygomycosis, typically inducing an eosinophilic granulomatous to pyogranulomatous inflammation affecting the dermis and subcutis.4 One important histologic feature that may help to differentiate lagenidiosis from pythiosis is hyphal diameter in tissue, which is typically larger and more variable for this Lagenidium pathogen (7 to 25 μm) than for P insidiosum (2 to 7 μm).1,4 The hyphal diameter in culture is also larger for Lagenidium spp (25 to 40 μm) than Pythium spp (4 to 10 μm).1,4 However, PCR assay or ribosomal DNA sequencing should be used for definitive differentiation of these agents.
Although cytologic and histologic findings may be supportive of lagenidiosis, a definitive diagnosis can be made on the basis of results of isolation of the organism via culture of fresh tissue followed by rRNA gene sequencing of the isolate. It should be noted that measurement of anti-Lagenidium antibody titers in serum samples from dogs by use of currently available ELISA or immunoblot techniques does not have sufficient sensitivity or specificity to be used as a basis for diagnosis.
Unfortunately, the prognosis associated with infection caused by this Lagenidium pathogen is poor to grave. Because lesions are often multifocal and extensive, complete excision is usually not possible. Even when cutaneous lesions may be resectable, the presence of occult lesions in the thorax or abdomen may make surgery futile. In addition, medical treatment has limited usefulness for the treatment of lagenidiosis or pythiosis because ergosterol, the target for most traditional antifungal drugs, is generally lacking in the oomycete cell membrane.4
References
1 Grooters AM, Hodgin EC, Bauer RW, et al. Clinicopathologic findings associated with Lagenidium sp infection in 6 dogs: initial description of an emerging oomycosis. J Vet Intern Med 2003; 17:636–646.
2 Kwon-Chung KJ. Phylogenetic spectrum of fungi that are pathogenic to humans. Clin Infect Dis 1994; 19(suppl 1):S1–S7.
3 Kerwin JL, Dritz DA, Washino RK. Pilot scale production and application in wildlife ponds of Lagenidium giganteum (Oomycetes: Lagenidiales). J Am Mosq Control Assoc 1994; 10:451–455.
4 Grooters AM. Pythiosis, lagenidiosis and zygomycosis in small animals. Vet Clin North Am Small Anim Pract 2003; 33:695–720.
