The oomycetes are a class of eukaryotes that resemble

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1 J Vet Intern Med 2003;17: Clinicopathologic Findings Associated with Lagenidium sp. Infection in 6 Dogs: Initial Description of an Emerging Oomycosis Amy M. Grooters, E. Clay Hodgin, Rudy W. Bauer, Carol J. Detrisac, Nadine R. Znajda, and Randall C. Thomas An oomycotic pathogen in the genus Lagenidium was isolated from tissues obtained from 6 dogs with progressive cutaneous disease. Initial clinical findings in 5 dogs included multifocal cutaneous lesions, subcutaneous lesions, or both associated with regional lymphadenopathy; the 6th dog initially was presented for evaluation of mandibular lymphadenopathy. Cutaneous lesions were ulcerated, exudative regions (often with necrosis and draining tracts) or multiple firm dermal or subcutaneous nodules. Two dogs subsequently developed hemoabdomen from great vessel rupture and died acutely. Four dogs were euthanized because of progression of subcutaneous lesions or lymphadenopathy. On postmortem examination, regional granulomatous lymphadenitis was found in all 6 dogs, great vessel invasion in 3 dogs, pulmonary lesions in 2 dogs, ureteral obstruction in 1 dog, mediastinal lymphadenitis in 1 dog, and hilar lymphadenitis with invasion of the distal esophagus and trachea in 1 dog. Histologically, lesions were similar to those associated with pythiosis and zygomycosis and were characterized by severe eosinophilic granulomatous inflammation (often with numerous large multinucleated giant cells) centered around broad (7 25 ), infrequently septate hyphae. Immunoblot analysis of the serologic response of 4 dogs to a soluble mycelial extract of Lagenidium giganteum indicated that each dog s serum recognized at least 10 different antigens of L giganteum. Culture of infected tissues yielded rapid growth of colorless to white submerged colonies. Microscopically, mature hyphae in culture were broad (25 40 ), segmented, and occasionally branching and produced motile laterally biflagellate zoospores in water culture. This report is the 1st description of infection caused by an oomycete other than Pythium insidiosum in any mammalian species. Key words: Aortic aneurysm; Canine; Cutaneous; Fungal; Lagenidiosis; Pythiosis; Vascular thrombosis. The oomycetes are a class of eukaryotes that resemble fungal organisms in morphology and in vitro growth characteristics but are phylogenetically distinct from true fungi. 1 A member of the kingdom Stramenopila (Chromista), the class Oomycetes includes many species that are economically important plant, fish, and crustacean pathogens, such as Phytophthora infestans (which causes late blight of potato, a disease best known for its role in the Great Irish Famine of 1845) and Saprolegnia ferax (a pathogen of hatchery-reared fish that also has been implicated in amphibian population declines around the world). 2 Pythium insidiosum, an aquatic oomycete initially identified in 1974 as the cause of swamp cancer in horses, 3 has long been considered to be the only mammalian pathogen in the class. 4 More recently, infections caused by P insidiosum have been recognized in a number of mammalian species. These infections typically are manifested by chronic progressive subcutaneous lesions in horses, dogs, cats, and cattle, and as severe segmental thickening of the gastrointestinal tract in dogs. 4,5 In human patients, P insidiosum infection, although rare, most often has been described as causing locally invasive subcutaneous periorbital or extremity From the Department of Veterinary Clinical Sciences, Louisiana State University, Baton Rouge, LA (Grooters); the Louisiana State Veterinary Medical Diagnostic Laboratory, Baton Rouge, LA (Hodgin, Bauer); and the Departments of Pathobiology (Detrisac) and Small Animal Clinical Sciences (Znajda, Thomas), College of Veterinary Medicine, University of Florida, Gainesville, FL. Preliminary results of this study were presented at the 10th Focus on Fungal Infections meeting, Atlanta, GA; March Reprint requests: Amy Grooters, DVM, Veterinary Clinical Sciences, Louisiana State University, Baton Rouge, LA ; agrooters@vetmed.lsu.edu. Submitted October 15, 2002; Revised December 26, 2002; Accepted February 4, Copyright 2003 by the American College of Veterinary Internal Medicine /03/ /$3.00/0 lesions, necrotizing arteritis associated with thrombosis, or intractable keratitis. 6 8 Animals and people living in the southeastern United States, as well as in other subtropical or tropical climates, are most frequently affected. The infective form of P insidiosum is thought to be the asexual reproductive structure, which is a motile biflagellate zoospore produced and released into water environments (probably in association with plant material). 9,10 The definitive diagnosis of pythiosis historically has been challenging. Although a presumptive diagnosis often is made on the basis of typical histologic findings (pyogranulomatous and eosinophilic inflammation surrounding broad, sparsely septate hyphae with nonparallel walls), these characteristics are shared by infections caused by zygomycete fungi in the order Entomophthorales, such as Conidiobolus coronatus and Basidiobolus ranarum. Because of the clinical and histologic similarities between pythiosis and zygomycosis, they often have been grouped together under the convenient but taxonomically obsolete term phycomycosis. Methods that have been used in the past for the diagnosis of pythiosis include culture of infected tissues, 11 immunohistochemical staining of tissue biopsies, 12 and immunodiffusion 13 or immunoblot 14 serology. More recently, assays that use enzyme-linked immunosorbent assay techniques 15 (for serologic evaluation) or molecular techniques 16 (for pathogen identification) have been developed. During the course of collecting culture, tissue, and serum samples from dogs with suspected pythiosis for use in the evaluation of these newly developed assays, we isolated and characterized a non-pythium oomycete pathogen with morphologic and molecular features consistent with its placement in the genus Lagenidium. 17 This report details the clinical, pathologic, and serologic findings associated with oomycosis in these dogs. A complete description and phylogenetic analysis of the newly identified Lagenidium species will be presented elsewhere.

2 638 Grooters et al Materials and Methods Microbiology For initial isolation, samples of infected tissue (3 5 mm 3 ) were placed directly onto the surface of vegetable extract (VE) agar, 18 peptone-yeast-glucose (PYG) agar, 19 or Sabouraud-dextrose agar a and incubated at 37 C. Isolates were subcultured by removing a small piece of agar from the edge of a 2 3-day-old colony and placing it directly onto PYG agar. In the long term, isolates were maintained at room temperature on PYG slants and subcultured every 3 months. Procedures for induction of asexual reproductive structures were modified from those previously described for P insidiosum and Lagenidium giganteum. For the 1st method, boiled grass blades were placed on the surface of a 2-day-old colony growing on 2% water agar and allowed to incubate at 37 C for 24 hours. The infected grass blades then were removed and placed into a 25-cm 2 tissue culture flask containing 25 ml of a dilute salt solution. 20 After incubation at 37 C for 24 hours, the edges of the infected grass blades were examined for production of zoosporangia with an inverted light microscope. For the 2nd method, 21 agar plugs (approximately 5 mm in diameter) were cut with a sterile cork borer from the edges of a 2-day-old colony growing on PYG agar that contained 10 ml/l sunflower oil. Several agar plugs with attached hyphae were placed into a 25-cm 2 tissue culture flask containing 25 ml of sterile distilled water. After incubation at 37 C for 24 hours, the edges of the agar plugs were examined for zoosporangia. Serology Western immunoblot analysis was performed to evaluate the ability of serum from dogs 3, 4, 5, and 6 to recognize antigens extracted from the following isolates: P insidiosum (ATCC b ), 6 C coronatus (ATCC 42064), 22 B ranarum (ATCC 24671), 23 L giganteum (ATCC 52675), 24 Lagenidium myophilum (ATCC 66280), and the Lagenidium species isolated from dog 1. Soluble mycelial antigens extracted from 5-day-old broth cultures as previously described 15 were electrophoretically separated on a 14% polyacrylamide gel 25 and then transferred to a nitrocellulose membrane with a submerged transfer unit. c Serum diluted at either 1 : 250 or 1 : 500 in phosphate-buffered saline (PBS) containing 5% dried nonfat milk was used to probe each membrane. Bound antibody was detected with HRP-conjugated rabbit anti-dog IgG, d developed with a chemiluminescent substrate e and visualized on radiographic film. Serum from a healthy dog (diluted at 1 : 250) was used as a negative control. Histology Formalin-fixed tissues were embedded in paraffin, sectioned at 3, and stained with hematoxylin and eosin (H&E) and Gomori s methenamine silver (GMS). Immunohistochemistry with a polyclonal antibody raised in rabbit against sonicated mycelial antigens of P insidiosum was performed on selected sections from each dog in which hyphae were visualized on GMS stains. This antibody previously was developed by the Louisiana State University Department of Pathology (Dr J. Newton) for diagnostic purposes. Although its specificity and sensitivity have not been documented in the veterinary literature, it has been used as a confirmatory test for pythiosis in a number of previously reported canine cases An avidin-biotin immunoperoxidase system was used, f with the anti-p insidiosum antibody as the primary antibody, biotinylated goat anti-rabbit IgG g as the secondary antibody, and a peroxidase substrate h for color development. Negative control slides (with preimmune antibodies as the primary antibody) and a positive control tissue (obtained from a dog with culture-confirmed pythiosis) were included with each immunohistochemistry procedure. Case Histories Dog 1 A 14-month-old intact male German Shepherd dog weighing 33 kg was referred to the Louisiana State University Veterinary Teaching Hospital (LSU-VTH) for evaluation of progressive skin disease of 3 months duration. The initial lesion was described by the owners as a 1 2-cm-diameter wound on the dorsal surface of the left tarsus that had progressed despite antibiotic therapy to multiple ulcerated lesions involving both hind limbs. It was not known whether the initial wound had been the result of trauma. Two months after the lesion was 1st noted, the dog developed lethargy, anorexia, and scrotal and bilateral hindlimb edema, which then had resolved after therapy with trimethoprim-sulfadiazine. The referring veterinarian had performed biopsies of multiple cutaneous lesions which disclosed severe pyogranulomatous inflammation associated with fungal structures. On physical examination at the LSU-VTH, areas of poorly circumscribed cellulitis and ulceration were observed that extended from just proximal to the tarsus to the distal metatarsal region on both hind limbs. These lesions contained areas of necrosis and granulation tissue, as well as multiple fistulous tracts from which a hemorrhagic mucopurulent exudate was easily expressed. Enlargement of both popliteal lymph nodes and the left inguinal lymph node also was present. A CBC disclosed hyperproteinemia, mild mature neutrophilia, monocytosis, and mild eosinophilia. Serum chemistry was within normal limits with the exception of hyperglobulinemia. Multiple biopsies of the cutaneous lesions were obtained under anesthesia. Examination of 1 biopsy specimen that was macerated and then digested in potassium hydroxide (KOH) for 30 minutes disclosed numerous broad, poorly septate, right angle branching hyphae. Histologic evaluation of the biopsy samples identified severe pyogranulomatous dermatitis with numerous broad (8 25 ), infrequently septate hyphae with irregular nonparallel walls. Zygomycosis was suspected because of the large hyphal diameter. Culture of a biopsy specimen resulted in the isolation of a fungal organism that initially was thought to be a zygomycete but later was identified as a member of the genus Lagenidium. Approximately 18 hours after recovery from anesthesia, the dog developed lethargy, fever, tachypnea, and signs of hypoperfusion. Despite aggressive fluid therapy, hypoperfusion progressed within 30 minutes to hypovolemic shock, cardiorespiratory arrest, and death. On postmortem examination, severe granulomatous dermatitis with intralesional hyphae, severe granulomatous lymphadenitis involving the popliteal, inguinal, iliac, and sacral lymph nodes (all with intralesional hyphae), and hemoabdomen were observed. A large hematoma extended along the ventral surface of the entire length of the lumbar spine, and a linear rupture of the caudal vena cava that appeared to be associated with compression or invasion by an enlarged sacral lymph node was identified. Lagenidium sp. was isolated from the iliac lymph node. Dog 2 A 2.5-year-old spayed female Jack Russell Terrier weighing 4.7 kg was referred to the University of Florida Veterinary Medical Teaching Hospital (UF-VMTH) for evaluation of progressive skin disease and lymphadenopathy of 1 month s duration. The owner initially had noted enlargement of both inguinal lymph nodes. Two weeks later, multifocal cutaneous nodules and draining tracts developed on the right and left flank and the right lateral thorax. Signs of systemic illness were not observed. The owner reported that the dog and its housemate swam frequently in a local lake. Biopsies of the cutaneous lesions performed by the referring veterinarian disclosed pyogranulomatous inflammation associated with broad, poorly septate hyphae. On physical examination at the UF-VMTH, cutaneous lesions in the following areas were identified: dorsal surface of right carpus (3 4 cm), right prescapular region (2 2 cm), left scapular region (3 3 cm), left ventral abdomen (4 4 cm), left flank (1 2 cm), and right flank (4 6 cm).

3 Lagenidium Infection in Dogs 639 Lesions were characterized by the presence of firm dermal nodules, erythema, alopecia, and multiple fistulous tracts, which drained a hemorrhagic purulent exudate. Bilateral inguinal lymphadenopathy also was present. A CBC, serum biochemistry, and urinalysis were within normal limits. Cytologic evaluation of an inguinal lymph node aspirate was nondiagnostic. Thoracic and abdominal radiographs were normal. Abdominal ultrasonography disclosed sublumbar lymphadenopathy. Biopsy of 2 of the skin lesions was performed. Cytologic examination of a tissue impression made from one of the biopsy samples identified pyogranulomatous inflammation with occasional nonseptate hyphae. Histopathologic evaluation identified pyogranulomatous dermatitis with intralesional hyphae. Culture of tissue biopsies yielded a fungal isolate that was thought to be a species of Basidiobolus. Treatment with itraconazole (10 mg/kg PO q24h) and enrofloxacin (5 mg/kg PO q12h) was initiated. In addition, over the next 2 weeks, the dog received increasing doses of amphotericin B lipid complex (0.5 2 mg/kg IV q48h) to a cumulative dose of 11 mg/kg. Fungal susceptibility testing by a broth microdilution method suggested resistance of the isolate to amphotericin B, itraconazole, fluconazole, and ketoconazole. Reevaluation 4 weeks after initiation of therapy indicated some improvement of the previously observed cutaneous lesions. However, new lesions had developed over the left shoulder and right biceps, and enlargement of the inguinal lymph nodes had progressed. Biopsy of the new cutaneous lesions indicated severe pyogranulomatous and eosinophilic dermatitis with few intralesional hyphae. Itraconazole was discontinued and treatment with potassium iodide (40 mg/kg PO q8h) and lufenuron (6.5 mg/kg PO q24hr) was initiated. Reexamination 4 weeks later revealed improving cutaneous lesions and resolution of the draining tracts. However, inguinal lymphadenopathy still was present. No signs of systemic illness had been observed. Abdominal ultrasonography indicated progressive sublumbar lymphadenopathy and unilateral hydronephrosis. Cytologic examination of a fine-needle aspirate of an enlarged sublumbar lymph node was suggestive of sarcoma. An excretory urogram identified unilateral hydronephrosis and hydroureter and a soft tissue mass in the caudal aspect of the retroperitoneal space. Exploratory laparotomy was performed because of a suspicion of retroperitoneal neoplasia causing ureteral obstruction, and a 10-cmlong sublumbar mass that had infiltrated both ureters at the level of the trigone was identified. The mass extended caudally into the pelvic canal and infiltrated the abdominal wall. Histopathologic examination of an ultrasound-guided needle biopsy sample of the sublumbar mass disclosed severe granulomatous inflammation with intralesional, rarely septate hyphae. After recovery from surgery, the dog was discharged on itraconazole (10 mg/kg PO q24h), lufenuron (10 mg/kg PO q24h), and terbinafine (13 mg/kg PO q24h). Reevaluation 6 weeks after surgery revealed resolution of the older cutaneous lesions but development of several new lesions that were draining. Abdominal ultrasonography at that time identified resolution of the hydronephrosis but continued sublumbar lymphadenopathy. Four weeks later, the dog presented for acute onset of abdominal discomfort and hind limb weakness. Abdominal ultrasonography disclosed a large caudal abdominal mass as well as a cystic structure in the dorsal abdomen caudal to the kidneys. Doppler examination indicated that this structure contained turbulent flow and suggested possible aortic aneurysm, which was confirmed by nonselective angiography. The dog died at home the next day. On postmortem examination, a rupture of the aortic aneurysm at the level of the renal arteries with hemorrhage into the peritoneal cavity and hypaxial musculature were observed. Adjacent to the rupture, the perivascular tissues were expanded by multiple granulomas, which entrapped fragments of lymphoid tissue and a portion of the ureter. The intimal surface of the aorta had a regional accumulation of fibrin and necrotic cellular debris and regional transmural granulomatous aortitis was present. This inflammatory process extended into the hypaxial musculature and associated adipose tissue. Mild unilateral hydronephrosis thought to be secondary to entrapment of the ureter in the inflammation also was present. All areas of inflammation contained multiple intracellular and extracellular broad hyphae with irregular septation. After postmortem examination, the isolate cultured from one of the original cutaneous lesions was submitted to one of the authors (AMG) for further evaluation and was determined on the basis of morphologic and molecular characteristics to be a Lagenidium species identical to that isolated from dog 1. Dog 3 Three months after the death of dog 2, her housemate (an unrelated 1.5-year-old spayed female Jack Russell Terrier weighing 8.8 kg) developed multiple dermal and subcutaneous nodules over the dorsum and ventrum. Surgical excision of cutaneous nodules over the sternum, 2nd mammary gland, and ventral surface of the left antebrachium was performed by the referring veterinarian. Histologic examination of these lesions indicated pyogranulomatous dermatitis with fungal structures, and the dog was referred for further evaluation. On physical examination at the UF-VMTH, multiple dermal and subcutaneous nodules were observed over the dorsal trunk, right prescapular region, inguinal mammary glands, and dorsal surface of the left antebrachium. The dermal lesions were firm, broad, plaquelike, and ranged in diameter from 1 to 3 cm. None of the lesions were ulcerated or draining. Mild eosinophilia was present on a CBC, but serum biochemistry was within normal limits. A punch biopsy of one of the subcutaneous lesions on the dorsal trunk was obtained for histopathology. In addition, fresh tissues and serum were submitted to one of the authors (AMG) for oomycete culture and immunoblot serology, respectively. Histopathologic examination identified severe pyogranulomatous panniculitis with intralesional bulbous to occasionally branching hyphal structures. Culture yielded an isolate that was morphologically identical to those obtained from dogs 1 and 2. Western immunoblot serology indicated that the dog s serum contained antibodies that recognized a large number of antigens of L giganteum, as well as those of the Lagenidium species isolated from dog 1 (Fig 1). Results of thoracic and abdominal radiography, as well as abdominal ultrasonography, were unremarkable except for the presence of microhepatica. Fasted and postprandial serum bile acid concentrations were within normal limits. Medical therapy was instituted with itraconazole (10 mg/kg PO q24h), terbinafine (14 mg/kg PO q24h), and lufenuron (5 mg/kg PO q24h). In addition, over the next 2.5 weeks, the dog received increasing doses of amphotericin B lipid complex (0.5 2 mg/kg IV q48h) to a cumulative dose of 10 mg/kg. During this time, the mammary and antebrachial lesions enlarged, several lesions in the interscapular area coalesced to form a large (10 20 cm) firm dermal to subcutaneous plaquelike lesion, and the dog developed moderate mandibular and superficial cervical lymphadenopathy. On reevaluation 3 weeks later, multiple cutaneous nodules had developed over the right stifle and left hock, and one of these lesions was ulcerated and draining. Topical therapy with amphotericin B deoxycholate (2.5 mg/ml in dimethyl sulfoxide (DMSO) applied to all lesions q48h) was initiated, and enrofloxacin (3 mg/kg PO q12h) was added to the oral medications. One month later, ulcerated and draining lesions were observed on both antebrachii, and the lesion over the right inguinal mammary gland had enlarged markedly. Topical therapy was continued with DMSO alone, and prednisone (1 mg/kg PO q24h) was added to decrease inflammation. Within 2 weeks, considerable reduction in size of the mammary and extremity lesions was noted, but new lesions had developed on the dorsum. Three weeks later, the dog was returned to the hospital with signs of discomfort, restlessness, shivering, exercise intolerance, and intermittent tachypnea. On physical examination, abscessation and serosanguineous drainage of a large subcutaneous lesion on the dorsum and marked engorgement of the superficial epigastric vessels were observed. Thoracic radiographs were unremarkable, but increased soft tissue opacity in the retroperitoneal space and cranial displacement of the kidneys were observed on abdominal radiographs. Abdominal ultrasonography identified marked thickening of

4 640 Grooters et al Fig 1. Immunoblot analysis demonstrating the ability of serum from dogs 3, 4, and 5 to recognize antigens of Pythium insidiosum (P. in), the canine pathogenic Lagenidium species isolated from dog 1 (L. sp), Lagenidium giganteum (L. gig), Lagenidium myophilum (L. myo), Basidiobolus ranarum (Bas), and Conidiobolus coronatus (Con). Markers on left indicate molecular weight in kilodaltons. the abdominal aorta and enlargement of the medial iliac and lumbar aortic lymph nodes. One week later, the owner elected to have the dog euthanized. On postmortem examination, multifocal severe granulomatous dermatitis, marked diffuse granulomatous inguinal lymphadenitis, and granulomatous peritoneal inflammations similar to that observed in dog 2 were identified. A 2-cm-diameter periaortic mass extended from 3 cm caudal to the diaphragm to the aortic bifurcation, and a 1.5-cmlong thrombus was present within the aorta. In addition, thrombi were observed in several small arteries within the site of inflammation. Histologically, the aortic lesion was characterized by disruption of the internal elastic lamina and virtually transmural extension of the granulomatous process. Irregularly branching hyphae with nonparallel walls and rare septa were present in sections of skin, lymph node, aorta, periaortic mass, and iliac arteries. Large clusters of hyphae were present within the aortic thrombus. Dog 4 A 1-year-old intact male Labrador Retriever was referred to the LSU-VTH for further evaluation of cutaneous lesions that had developed over an unknown period of time. The owners had noted that the dog recently had begun losing weight. The referring veterinarian had performed biopsies of cutaneous lesions involving the right elbow and right medial thigh, which disclosed eosinophilic and pyogranulomatous inflammation centered around broad, variably septate fungal hyphae. On physical examination at the LSU-VTH, multiple firm, irregular dermal nodules (ranging in diameter from 1 or 2 mm up to 10 mm) were observed on the caudal aspect of the right elbow and the caudal portion of the right medial thigh. Western immunoblot serology indicated that the dog s serum contained antibodies that recognized antigens extracted from the Lagenidium sp. isolated from dog 1, as well as those extracted from L giganteum (Fig 1). The owners elected euthanasia because of the guarded prognosis. Postmortem examination identified severe eosinophilic granulomatous dermatitis and panniculitis involving the right elbow and right medial thigh, severe eosinophilic granulomatous lymphadenitis involving the right axillary and inguinal lymph nodes, and localized eosinophilic granulomatous pneumonia, which appeared grossly as a large (7 5 3 cm) solid mass involving the left mainstem bronchus and surrounding pulmonary parenchyma (Fig 2). Hyphal organisms were detected histologically within the skin, lymph node, and pulmonary lesions, and Lagenidium sp. was isolated from all 3 tissues. Dog 5 Fig 2. Gross photograph of the tracheal bifurcation of dog 4. There is a mass (arrows) surrounding the left mainstem bronchus and invading the adjacent pulmonary parenchyma. Histologically, this mass was characterized by eosinophilic granulomatous inflammation with intralesional hyphae. Tr, trachea. Bar 2 cm. A 6-year-old female spayed Springer Spaniel was presented to the LSU-VTH for euthanasia after a 3-month course of progressive skin disease. The owners 1st had noted several masses on the skin of the dog s ventral abdomen. They also reported that the dog swam daily in a local lake. Physical examination by the referring veterinarian shortly thereafter identified a large (5 cm diameter), firm, raised lesion with an ulcerated center on the ventral abdomen; a smaller (2 cm diameter), firm, raised lesion also on the ventral abdomen; and a 1.5-cm-diameter, firm, raised lesion on the medial aspect of the right carpus. Cytologic evaluation of a fine needle aspirate of the largest lesion disclosed neutrophilic inflammation. After treatment with cephalexin for 3 weeks, the largest lesion had become more extensive, and a raised firm area of alopecia had developed on the dorsal aspect of the left carpus. Surgical resection of the largest mass was performed, and the mass was found to be firmly adhered to the underlying abdominal wall. Histologic evaluation indicated marked diffuse granulomatous inflammation with areas of necrosis that contained funguslike structures. Over the next 4 weeks, the lesions became more extensive despite treatment with cephalexin (25 mg/kg PO q12h). Therapy with itraconazole (5 mg/kg PO q24h) and amoxicillin-clavulonic acid (18 mg/kg

5 Lagenidium Infection in Dogs 641 focally extensive granulomatous esophagitis, granulomatous tracheitis with focal epithelial disruption, and multifocal necrotizing pulmonary granulomas. All lesions contained numerous broad hyphal structures with thick nonparallel walls and right angle branching. Lagenidium sp. was isolated from samples of lung and lymph nodes. Fig 3. Gross photograph of a large, raised, ulcerated and exudative cutaneous lesion on the ventral abdomen of dog 5. Histologic examination of this lesion revealed severe ulcerative granulomatous dermatitis with intralesional hyphae. Bar 2 cm. PO q12h) was initiated. Two weeks later, some of the lesions had improved, but others had undergone more extensive necrosis and ulceration. One month after itraconazole therapy had been initiated, the dog became lethargic and the owners elected referral for euthanasia and postmortem examination. On physical examination at the LSU-VTH, 2 large (9 9cmand 4 9 cm), raised, ulcerated, exudative cutaneous lesions were observed on the ventral abdomen (Fig 3), one with associated draining tracts. In addition, a raised, firm, ulcerated cutaneous lesion (3 5 cm) was found on the dorsal aspect of each carpus. Inguinal lymphadenopathy also was present. On postmortem examination, severe ulcerative granulomatous dermatitis and panniculitis, as well as severe granulomatous inguinal and mediastinal lymphadenitis, were identified, and all lesions contained intralesional hyphae. Lagenidium sp. was isolated from the large cutaneous lesion, as well as from the inguinal and mediastinal lymph nodes. Western immunoblot serology indicated the presence of antibodies that recognized antigens extracted from the Lagenidium sp. isolated from dog 1 and those extracted from L giganteum (Fig 1). Dog 6 A 2-year-old spayed female mixed breed dog was referred for euthanasia and postmortem examination after a 2-month course of progressive mandibular lymphadenopathy. A biopsy specimen of one of the mandibular lymph nodes by the referring veterinarian had contained histologic features consistent with pythiosis. Shortly thereafter, the dog had developed dyspnea thought to be related to progressive lymph node enlargement, and the referring veterinarian had performed a partial surgical excision of the right mandibular lymph node. A portion of the lymph node was submitted to one of the authors (AMG) for culture, and an oomycete consistent with Lagenidium sp. was isolated. The dog s condition continued to deteriorate, and the owners elected referral for euthanasia and postmortem examination. On physical examination at the LSU-VTH, moderate to severe bilateral mandibular lymphadenopathy was found. On postmortem examination, a mass cm involving the right mandibular lymph node, a mass cm involving the left mandibular lymph node, and a mass cm at the pulmonary hilus that extended around the carina and invaded the distal trachea and esophagus were identified. In addition, the parenchyma of the left caudal lung lobe was firm and contained numerous 2 5-mm-diameter nodules. Histopathologic evaluation identified severe bilateral granulomatous mandibular lymphadenitis, granulomatous hilar lymphadenitis, Results Microbiology Culture of tissue samples from each dog yielded rapid growth ( 5 mm/d on PYG agar at 37 C) of a colorless to white submerged colony. Growth on PYG agar was subjectively judged to be heavier than that on either VE or Sabouraud/dextrose agar. Microscopically, mature hyphae on PYG were broad (25 40 ), hyaline, segmented, and frequently branching, with branches that were perpendicular to and often as wide as the main hyphae. Occasionally, short branches appeared as budding protuberances emerging from segments of the main hyphae. Internal structures often were visualized within hyphal segments. Zoosporulation was successfully induced in isolates from dogs 4, 5, and 6 by the grass blade method. After incubation of infected grass blades in induction medium for 24 hours, motile biflagellate zoospores could be seen erupting from terminal vesicles that had developed from tubular zoosporangia extending from the grass blade edges. Zoospores were not produced by any of the isolates by the agar plug method. Serology Immunoblot analysis performed with sera from dogs 3, 4, 5, and 6 determined that each dog s serum contained antibodies that recognized at least 10 antigens of L giganteum, as well as at least 10 antigens of the pathogenic Lagenidium species isolated from dog 1 (Fig 1). At least 5 antigens recognized by each dog s serum appeared to be shared between L giganteum and the Lagenidium species pathogenic for dogs. Serum from each of these dogs also recognized between 1 and 7 antigens of P insidiosum, as well as between 0 and 7 antigens of L. myophilum. None of the sera tested recognized antigens of B ranarum or C coronatus. Serum from the healthy dog did not recognize any of the antigens tested. Histopathology The histologic lesions in skin were most commonly characterized by eosinophilic granulomatous inflammation of the dermis and subcutis (Fig 4). In some dogs, eosinophils were a prominent inflammatory cell type, whereas in others, coalescing to diffuse pyogranulomatous inflammation with fewer eosinophils was observed. Areas of necrosis and suppuration were sometimes present. Multinucleated giant cells often were observed and in some lesions were numerous. Plasma cells often were present and tended to surround discrete eosinophilic granulomas. In skin and all other infected tissues, hyphal structures usually were recognizable on H&E-stained sections by a visible cell wall and occasional internal structures (Fig 5A). The hyphae could be found intracellularly within giant cells (Fig 5B), as well as extracellularly within areas of eosinophilic inflammation, neu-

6 642 Grooters et al Fig 4. Photomicrograph of a cutaneous lesion on the hind limb of dog 1, showing severe multifocal to coalescing pyogranulomatous inflammation of the dermis and subcutis. Hematoxylin and eosin stain. Bar 200. Fig 5. Photomicrographs showing Lagenidium sp. hyphae in tissue. (A) Extracellular hyphae with visible cell wall (arrows) within infected lung tissue from dog 6; (B) intracellular hyphae within giant cell (arrow) in infected lymph node from dog 5. Hematoxylin and eosin stain. Bar 20. trophilic inflammation, or necrosis. On GMS-stained sections, numerous broad, thick-walled, irregularly septate hyphae of varying diameter were easily visible (Fig 6A). In some sections, hyphae appeared as round or bulbous structures (Fig 6B). Right angle branching occasionally was observed. Hyphal diameter ranged from 7 to 25, with a mean diameter of 12. A scant to thin granular eosinophilic sleeve (which appeared to be caused by degranulating eosinophils) was noted around hyphae in some tissues. In lymph node sections, lymphadenitis characterized by partial to extensive effacement of lymphoid tissue by coalescing granulomatous inflammation with giant cells and areas of necrosis was observed. Fewer foci of neutrophils or eosinophils also were present. Hyphae were present in giant cells and within areas of necrosis. Perinodal granulomatous inflammation also was present and often involved lymphatics and arteries (Fig 6A). In some dogs, partial to total effacement or obstruction of arterioles was noted. In sections of lung, diffuse suppurative to pyogranulomatous inflammation with numerous giant cells was present and effaced bronchial submucosa and surrounding parenchyma. Hyphal organisms were present both intracellularly and extracellularly. Lymphangitis also was observed and was characterized by similar inflammation that disrupted the lymphatic walls at multiple sites and protruded into the lumina. In the dog with the hilar mass (dog 6), granulomatous inflammation appeared to extend from the hilar lymph node into the distal trachea, esophagus, and surrounding pulmonary parenchyma. In the trachea, the inflammation invaded between tracheal rings, involving the submucosa and resulting in focal mucosal disruption. In the esophagus, effacement of serosal and muscular layers and extension of inflammation into the submucosa was observed. Great vessel lesions were characterized by regional transmural granulomatous vasculitis associated with perivascular inflammation. In the aorta of dog 3, large numbers of hyphae were present within the lumen and remnants of the intima, with rare hyphae in the media. Hyphae did not align along any structure, including elastin, within arterial walls. Anti-P insidiosum Immunohistochemistry Immunohistochemical evaluation with a polyclonal antibody raised against antigens of P insidiosum identified weak positive staining of hyphae in all tissues evaluated.

7 Lagenidium Infection in Dogs 643 Fig 6. Photomicrographs of tissue sections stained with Gomori s methenamine silver. (A) Broad, thick-walled, irregularly septate hyphae associated with granulomatous vasculitis in a lymph node from dog 6; (B) numerous hyphae of varying diameter (some of which have a round or bulbous appearance) in an infected lymph node from dog 5. Bar 20. The intensity of this staining was considerably less intense than that of the positive control (tissue containing P insidiosum hyphae). Discussion The genus Lagenidium comprises more than 50 species, most of which occur as parasites of algae, fungi, rotifers, nematodes, crustaceans, Daphne, and mosquito larvae. The most well-studied species, L giganteum, is a facultative pathogen of mosquito larvae that is currently approved by the Environmental Protection Agency for use in the United States as a biological control agent for mosquito populations. 29,30 These data suggest that the Lagenidium species pathogenic to dogs is closely related to L giganteum on the basis of strong antigenic similarities (demonstrated by the degree to which sera from Lagenidium-infected dogs recognize antigens of L giganteum), as well as molecular similarities. 17 However, differences in their in vitro growth characteristics (Grooters, unpublished observations), as well as the failure of L giganteum to infect rodents in mammalian safety studies, suggest that the Lagenidium species pathogenic to dogs and L giganteum are likely distinct species. The clinicopathologic features of lagenidiosis manifested by the dogs of this report are similar in many aspects to those that previously have been associated with cutaneous pythiosis. 5,31,32 All 6 affected animals were young to middleaged dogs living in the southeastern United States, and at least 3 swam frequently in a local lake or pond. Five of the 6 dogs initially were presented for evaluation of progressive cutaneous or subcutaneous lesions involving the extremities or trunk. Grossly, these lesions appeared as firm dermal or subcutaneous nodules or as ulcerated, thickened, edematous areas with regions of necrosis and numerous draining tracts from which a hemorrhagic mucopurulent exudate was easily expressed. Regional lymphadenopathy was noted on physical examination in 4 of the 5 dogs that presented with cutaneous lesions and was the initial complaint in dog 6 (in which cutaneous lesions were not observed). Similar to the clinical course typically associated with cutaneous pythiosis, the skin lesions in dogs with lagenidiosis tended to be progressive, locally invasive, and unresponsive to either surgical excision or aggressive systemic antifungal therapy, including itraconazole and amphotericin B lipid complex. Despite these similarities, a number of important differences between canine cutaneous pythiosis and lagenidiosis should be noted. Perhaps the most striking difference is the frequency with which lesions in organs other than skin and regional lymph nodes were noted in the Lagenidium-infected dogs described here. Distant sites in which inflammatory lesions containing hyphae were observed included lung (2 dogs), aorta (2 dogs), caudal vena cava (1 dog), esophagus, trachea, hilar lymph node (1 dog), and mediastinal lymph node (1 dog). In addition, cutaneous lesions were absent in 1 dog (dog 6) that was initially presented for evaluation of mandibular lymphadenopathy. In contrast, the presence of lesions in organs other than skin and regional lymph nodes is rare among dogs with cutaneous pythiosis. 5,11,31 34 These findings raise important questions about potential routes of infection and modes of systemic dissemination in dogs with lagenidiosis. Because the motile zoospore of P insidiosum is attracted to animal hair as well as to cut areas of animal skin, 10 it has been hypothesized that cutaneous pythiosis occurs when zoospores encyst in and invade areas of broken skin. 9,34 A similar mechanism could be responsible for the production of cutaneous lesions in dogs with lagenidiosis, but the frequent development of lesions in distant organs suggests that hematogenous or lymphatic dissemination of infection might occur more often in dogs with lagenidiosis than in those with pythiosis. In addition, the distribution of lesions in dog 6 (in which lesions were not observed in skin but were present in the pulmonary parenchyma and hilar lymph node) suggests that the pathogen might have been inhaled or aspirated. Although pulmonary parenchymal infection has been reported as a sequela to subcutaneous pythiosis in a small number of horses, 35,36 primary pulmonary pythiosis has not been reported in any companion animal species. The great vessel invasion observed in 3 of the 6 Lagenidium-infected dogs is an interesting finding in light of previous reports of arteritis attributed to P insidiosum infection in thalassemic patients in Thailand. 7,37,38 The vascular le-

8 644 Grooters et al sions and associated thrombosis in these human patients, although most often involving arteries of the extremities, are similar to the great vessel lesions observed in the Lagenidium-infected dogs. Thrombosis and, rarely, arteritis have been described in dogs with cutaneous pythiosis 31 and also occur in dogs with gastrointestinal pythiosis (especially those with lesions involving the mesenteric root). 39 Both intestinal infarction 39,40 and hemoabdomen secondary to rupture of a mesenteric vessel (Grooters, unpublished observations) have been associated with gastrointestinal pythiosis in dogs. However, invasion or rupture of the aorta or caudal vena cava has not been reported in dogs with either cutaneous or gastrointestinal pythiosis. Histologically, the lesions associated with Lagenidium infection resemble those previously described in animals with pythiosis and zygomycosis. All 3 diseases produce pyogranulomatous and eosinophilic inflammation centered around broad, infrequently septate hyphae with nonparallel walls and right angle branching. For this reason, routine histopathologic evaluation alone is insufficient for the definitive diagnosis of pythiosis, zygomycosis, or lagenidiosis. However, we did note some subtle differences between the histologic characteristics observed in the Lagenidium-infected dogs and those previously described in animals with pythiosis and zygomycosis. First, the cell wall (and occasionally the internal structures as well) of Lagenidium sp. hyphae usually were visible on H&E-stained sections (Fig 5). In contrast, the hyphae of P insidiosum, C coronatus, and B ranarum rarely are visualized on H&E-stained sections, although hyphal ghosts can be apparent within granulomas as clear spaces delineated by a band of acellular eosinophilic material (ie, eosinophilic sleeve) surrounding the hyphal wall. 39,41 43 Another distinguishing histologic feature noted was the large and variable hyphal diameter, as measured on GMS-stained sections. The diameter of Lagenidium hyphae observed in the dogs of this report (mean, 12 ; range, 7 25 ) is significantly larger than that reported for P insidiosum (mean, 4 ; range, 2 7 ), Conidiobolus spp. (mean, 8 ; range, 5 13 ), or Basidiobolus spp. (mean, 9 ; range, 5 20 ). 43 Lagenidium hyphae were further distinguished from those of the Entomophthorales by the absence of a thick eosinophilic sleeve, which often is as wide as 5 10 for Conidiobolus spp. and up to 25 for Basidiobolus spp. 43 In contrast, the eosinophilic sleeve around Lagenidium sp. hyphae, when present, was scant to thin. Although none of these histologic features are consistent and distinct enough to be used as the sole basis for definitive differentiation of lagenidiosis from pythiosis and zygomycosis, they might provide the pathologist with an index of suspicion that would prompt further evaluation for lagenidiosis. The diagnostic test of choice for lagenidiosis in veterinary patients is culture of infected tissues followed by both morphologic and molecular identification of the pathogen. Isolation is most successful if small pieces of fresh tissue are placed directly on the surface of PYG agar and incubated at 37 C; hyphal growth usually is apparent within 24 hours. Samples that must be submitted to an outside laboratory should be shipped at ambient temperature to arrive within 48 hours. Isolation of either Lagenidium sp. or P insidiosum from samples with bacterial contamination is often more successful when ampicillin (100 g/ml) and streptomycin (200 g/ml) are incorporated into the agar. 44 Morphologic features of the Lagenidium species pathogenic to dogs that help differentiate it from P insidiosum include its large hyphal diameter, its propensity to form oval to round hyphal segments, and the ease with which its internal structures can be visualized. The production of laterally biflagellate motile zoospores confirms the identity of an isolate as an oomycete but does not differentiate Lagenidium sp. from P insidiosum. For molecular confirmation of identity, a PCR-based assay that differentiates the canine Lagenidium pathogen from P insidiosum, as well as C coronatus and B ranarum but not L giganteum, has been developed. 45 This assay has been applied successfully to DNA extracted from either cultured isolates or infected tissues. 46 Similar molecular tools have been developed for the identification of P insidiosum. 16 The serology data in the 6 dogs presented here suggest that immunoblot analysis also could be a useful tool for the diagnosis of lagenidiosis. However, the degree to which sera from dogs with pythiosis, zygomycosis, or other systemic mycoses recognize Lagenidium antigens must be more extensively evaluated before the specificity of immunoblot analysis for the diagnosis of lagenidiosis can be estimated. In addition, a large number of sera from healthy dogs should be evaluated for potential anti-lagenidium activity due to previous exposure to nonpathogenic Lagenidium species. Similarly, estimates of sensitivity will require the evaluation of a greater number of sera from Lagenidium-infected dogs. In the immunohistochemical evaluations, the weak positive staining of Lagenidium hyphae by the anti-p insidiosum antibody suggests that these 2 genera have some antigenic similarities. This finding is not surprising given their close phylogenetic relationship and suggests that the specificity of this previously utilized anti-p insidiosum immunohistochemical antibody might be inadequate for the differentiation of pythiosis from lagenidiosis. Although the difference in staining intensity between Lagenidium and P insidiosum hyphae with this antibody is marked, these findings suggest that positive anti-p insidiosum immunohistochemistry results should be viewed with caution unless the antibody s specificity has been documented. This precaution is especially important given that positive staining with this same anti-p insidiosum antibody has been used as the basis for the definitive diagnosis of pythiosis in a number of previous reports in the veterinary literature A P insidiosum specific polyclonal antibody that does not stain Lagenidium hyphae has been developed by the authors, and its use in an immunohistochemical assay is currently under evaluation. 47 Results of this study suggest that lagenidiosis should be considered as a differential diagnosis in any patient with clinical or histological findings suggestive of pythiosis or zygomycosis. Although some authors have chosen to base the diagnosis of pythiosis on routine histologic findings alone, 32 our experience, as well as that of other authors, 33,42 suggests that the histopathologic characteristics of pythiosis, lagenidiosis, and zygomycosis are insufficiently unique to justify their use as the sole basis for a definitive diagnosis. The recent development of highly specific serologic 15

9 Lagenidium Infection in Dogs 645 and molecular 16,45 tools for the differentiation of these infections will provide veterinarians with the opportunity to make a definitive diagnosis, even when culture is not possible or has been unsuccessful. However, the identification of a 2nd mammalian pathogen in the class Oomycetes underscores the importance of obtaining a culture-based diagnosis (preferably with molecular confirmation of the pathogen s identity) whenever possible. Not only will this approach be important for providing appropriate treatment recommendations and accurate information about prognosis, it also will be critical for the future identification of previously unrecognized oomycotic and zygomycotic pathogens and for better characterization of the epidemiology of these devastating infections. Footnotes a Sabouraud dextrose agar, BD Diagnostic Systems, Sparks, MD b American Type Culture Collection, Manassas, VA c Mini Trans-Blot Transfer Cell, Bio-Rad Laboratories, Hercules, CA d HRP-conjugated rabbit anti-dog IgG, Rockland Immunochemicals, Gilbertsville, PA e ECL Western Blotting Detection Reagents, Amersham Pharmacia Biotech, Piscataway, NJ f Vectastain Elite ABC kit, Vector Laboratories, Burlingame, CA g Anti-rabbit IgG (H L), Vector Laboratories, Burlingame, CA h Vector NovaRed substrate, Vector Laboratories, Burlingame, CA Acknowledgments The authors thank Drs Tom Vicek, Doo-Youn Cho, Sundeep Chandra, Elizabeth Uhl, William Castleman, Calvin Johnson, Pamela Ginn, Rose Raskin, Kristen Henson, and Ron Snider for assistance with pathologic evaluations and Drs Nola Lester, Susan Newell, and Lisa Ferretti for radiographic examinations. Dr Grooters work is supported by grants from the Morris Animal Foundation and the American Academy of Veterinary Dermatology. References 1. 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