Oomycosis: Pythiosis in the Dog, Horse and Cat and Lagenidiosis in the Dog

Bradley A. Stephens, DVM; Heather L. Tarpley, DVM; Pauline M. Rakich, DVM, PhD; Kenneth S. Latimer, DVM, PhD; Melanie E. Johnson, DVM; Perry J. Bain, DVM, PhD

Class of 2005 (Stephens), Department of Pathology (Tarpley, Johnson, Bain, Latimer), and Athens Veterinary Diagnostic Laboratory (Rakich), College of Veterinary Medicine, University of Georgia, Athens, GA 30602-7388

Introduction

Pythiosis is an uncommon cutaneous/subcutaneous, gastrointestinal, or multisystemic disease caused by a water mold. It has been reported in dogs, horses, cats, cattle, humans and a captive zoo bear.¹⁰ Cases have predominantly been documented worldwide in tropical, subtropical and other wet, warm climates. Recent emergence of pythiosis in more temperate regions suggests that the disease is more widespread than previously suspected. Horses are most commonly affected followed by dogs and, rarely, cats.

Lagenidiosis is an emerging cutaneous/subcutaneous or multisystemic disease that has been recently reported in dogs from the southern United States. Lagenidiosis also is caused by a water mold and is clinically very similar to the cutaneous form of pythiosis. However, multiorgan involvement occurs more frequently in lagenidiosis.⁴

The recent discovery of infection in dogs with Lagenidium sp., as well as the clinical resemblance of lagenidiosis and pythiosis to the zygomycoses (once collectively referred to as "phycomycosis"), have made timely diagnoses of oomycosis difficult and have increased the demand for more rapid and accurate diagnostic tests.

Etiology and Biology

Pythiosis is caused by the aquatic pathogen Pythium insidiosum in every reported case of mammalian disease.¹⁰ There are nearly eighty five species of Pythium, most of which are parasites of plants, fish, and crustaceans.¹⁰ An as yet undetermined species of Lagenidium is responsible for lagenidiosis in dogs.⁴ The genus Lagenidium includes more than 50 species of which most are natural parasites of algae, fungi, rotifers, nematodes, crustaceans, Daphne, and mosquito larvae.⁴ L. giganteum, a parasite of mosquito larvae, has been approved by the Environmental Protection Agency for use in the United States as a biological control of the adult mosquito population.⁴

Both Pythium and Lagenidium species belong to the kingdom stramenopila, phylum oomycota, and class oomycetes.¹⁰ The oomycetes, often referred to as water molds, have many characteristics in common with the fungi but are phylogenetically distinct.³ Oomycetes differ from true fungi in that they produce motile, flagellate zoospores, have a cell wall that does not contain chitin, and have different metabolic pathways and organelle structure. The lack of sterol components within the plasma membrane of oomycetes is clinically significant since ergosterol is the target molecule for the action of most antifungal drugs.³

Pythium sp. and Lagenidium sp. are believed to have life cycles that are similar to that of other oomycetes (Figure 1).¹⁰ When placed in water, oomycete hyphae develop sporangia which eventually form multiple biflagellate zoospores. The zoospores then break through the vesicle wall and swim away and encyst. These zoospores are thought to be the infectious stage and have been shown to display chemotactic behavior toward damaged animal and plant tissues. Oomycetes also have the ability to form resistant spores when ambient conditions do not favor zoospore formation.¹⁰

Figure 1. General depiction of oomycete life cycle.11 Larger Image >>

Epidemiology

Pythiosis has been reported in Australia, Brazil, Burma, Colombia, Costa Rica, Indonesia, Japan, New Guinea, Thailand, and the United States. Within the United States, cases of pythiosis have been reported in Alabama, Florida, Georgia, Illinois, Indiana, Louisiana, Mississippi, Missouri, New York, New Jersey, Oklahoma, South Carolina, Tennessee, and Wisconsin.^3,8,10

Lagenidiosis occurs in roughly the same geographic areas as pythiosis and cases of disease have been reported from Florida, Louisiana, Tennessee, Texas, and Virginia.⁴

Young, male, large-breed dogs are most often affected with reports of predilection in the German Shepherd Dog.³ Regardless of species and age, animals exposed to warm, standing fresh water are more likely to be in contact with the infectious zoospore and may have increased risk for disease. Reports of animals being infected with no known history of being near water suggests that animals can be inoculated by contact with resistant spores that form in wet soil and on grass.² Although it is unknown if zoospores are capable of penetrating intact skin, Pythium insidiosum and Lagenidium sp. are considered to be true pathogens because immune dysfunction has not been observed in affected animals.¹⁰

Pathogenesis

Oomycetes cause disease when animals with damaged skin come into contact with zoospores, which encyst in the exposed tissue.¹⁰ The zoospores then produce hyphae which cause disease by mechanically penetrating the surrounding tissue.¹⁰ In cases of gastrointestinal disease in dogs, infection is thought to develop via ingestion of the organism which encysts and invades the damaged mucosa of the alimentary tract.⁷ In widespread disease, the organism usually penetrates the gastrointestinal tract and disseminates to other intra-abdominal organs. It has also been speculated that thrombosis, arterial disruption, and the intense inflammatory response associated with invasion of the organisms may allow the disease to spread to other tissues. Even with treatment, most cases are fatal.^8,9,10

Clinical Findings

Equine pythiosis - Infections in horses caused by Pythium insidiosum are most commonly restricted to the cutaneous and subcutaneous tissues (Fig. 2).^8,9 There may be single or multiple nonhealing, rapidly enlarging, tumor-like, nodular masses with multiple draining tracts and serosanguineous discharge.¹⁰ These lesions are usually on the limbs and ventral abdomen but occur anywhere on the body. The inciting wound is often ulcerated.^8,9,10 The lesions are usually intensely pruritic and horses may mutilate the wounds if not closely monitored.^8,9,10 There may be mild to marked lymphadenopathy. In chronic infections (>4 weeks duration), Pythium sp. may spread to the underlying bone and cause lameness.⁹ Skin lesions often contain "kunkers," yellowish gritty coral-like bodies ranging from 0.5 to 1.5 cm in diameter (Fig. 3). Kunkers are composed of necrotic eosinophils, Pythium sp. hyphae, and necrotic vessels; however, they are not specific for pythiosis.^8,9

Figure 2. Draining mass on the rear leg of a horse with Pythium insidiosum infection (images courtesy of Noah’s Arkive, University of Georgia).

Figure 3. Section of a skin biopsy from a cutaneous mass of a horse with pythiosis. Notice the granular, irregulary-shaped, yellow "kunkers" within the diseased skin (image courtesy of Noah’s Arkive, University of Georgia).

Horses rarely develop gastrointestinal disease similar to that occurring in dogs. Horses with gastrointestinal pythiosis may have signs of gastrointestinal obstruction, weight loss, anorexia, diarrhea, and colic. In other individuals, gastrointestinal disease may be unaccompanied by signs of systemic illness.¹⁰

Following initial infection, the organism may disseminate to distant areas through the lymphatics. The lymph nodes, bones, or lungs may be involved.¹⁰

In laboratory testing, the complete blood cell count and biochemical profile are often unremarkable. However, leukocytosis with mature neutrophilia and eosinophilia has been reported in some cases. A normocytic, normochromic anemia often develops due to the chronic inflammation or excessive self-mutilation and blood loss.^8,9,10

Canine gastrointestinal pythiosis - Canine pythiosis most commonly presents as gastrointestinal disease (Fig. 4).³ Affected dogs often will have a history of upper gastrointestinal tract obstruction and may have a palpable abdominal mass.³ Any part of the gastrointestinal tract may be affected with chronic granulomatous enteritis; clinical signs reflect which region of the alimentary tract is diseased.³ Usual clinical signs include chronic intractable diarrhea, vomiting, anorexia, depression, and chronic weight loss.³ The stomach and duodenum are the most common sights of infection.³ Stomach lesions may be accompanied by abdominal pain and "coffee ground" vomitus due to ulceration and gastric bleeding.^3,7,10 With distal small intestinal disease, chronic diarrhea is more common.³ With colonic involvement and ulceration there may be bloody diarrhea; either hematochezia (fresh blood) or melena (digested blood) depending on the region of ulceration.³ Oropharyngeal lesions have been reported but are much less common. Chronic esophagitis may evolve into megaesophagus or cause hypersalivation.³ Occasionally, infection is found only in the mesenteric lymph nodes.³ Dogs, like horses, will not show signs of systemic disease unless intestinal obstruction, infarction, or perforation are present.^3,10

Figure 4. Intestinal mass in a dog with oomycete infection (image courtesy of Noah’s Arkive, University of Georgia).

In most cases of gastrointestinal pythiosis, hematologic and serum chemistry findings are unremarkable. Mild to moderate nonregenerative anemia, neutrophilic and eosinophilic leukocytosis, and panhypoproteinemia may be observed occasionally.^3,7,10 Clinical presentation of patients with gastrointestinal pythiosis is often late in the course of disease due to the absence of clinical signs and abnormal laboratory test results.³

Canine cutaneous/subcutaneous pythiosis - The cutaneous/subcutaneous form of pythiosis is less frequent in dogs than the gastrointestinal form of disease.³ As in horses, a predisposing wound usually allows infiltration of the infective zoospores.² Many animals have a history of swimming or being in "swampy" areas. The German Shepherd Dog may be predisposed.² Non-healing, tumor-like nodules with ulceration and draining tracts may be present. The discharge from these lesions is serosanguineous to purulent.^2,3 These lesions usually are non-pruritic to slightly pruritic and often enlarge rapidly.^2,3 Some lesions may have foci of necrosis and a spongy appearance.³ Such lesions are usually on the limbs, thoracic wall, tail head, or perineal region but can be seen anywhere.^2,3,10 The cutaneous form of pythiosis is rarely complicated by gastrointestinal involvement. Eosinophilia is the most common abnormality on the complete blood cell count; however, laboratory data are usually within the reference interval.^2,3,10

Canine lagenidiosis - The clinical presentation of canine lagenidiosis is nearly identical to that of the cutaneous form of pythiosis. The tumor like masses of lagenidiosis are identical in appearance to those of pythiosis and commonly affect the limbs, mammary glands, trunk, or vulva. The notable difference in these diseases is that lagenidiosis disseminates to other organs much more commonly. Spontaneous dissemination of disease may involve the lungs, aorta, cranial mediastinum, and lymph nodes.⁴

Canine disseminated pythiosis: Disseminated or metastatic pythiosis occurs rarely in dogs.³ Multiple organs may become involved following the gastrointestinal or cutaneous form of disease. A dog with prostatic pythiosis has been reported which had no apparent subsequent gastrointestinal or cutaneous disease.⁶ It is thought that systemic corticosteroid use may have lead to dissemination of the infection.³ Another possible mechanism for dissemination may be generalized immunosuppression from stress or another disease process.

Feline pythiosis - Pythium insidiosum infection in cats is rare. Cases that have been reported are usually of a subcutaneous infection. These lesions are much less invasive and aggressive than those seen in dogs.^1,10 Infection is confined to the subcutaneous region and lacks draining tracts that are usually seen in horses and dogs.¹⁰ Nodular masses may be observed on the limbs, abdomen, and inguinal region. In one cat with pythiosis, lesions were observed within the nasal cavity and nasopharynx and extended into both orbital cavities.¹ This case apparently is the only report of retrobulbar pythiosis.

Diagnostic Imaging

In most cases of oomycosis, imaging studies are of little value as the findings are not specific and often inconsistent.^6,8,9 Radiographs may show areas of soft tissue swelling or bony lysis and periosteal reaction.⁹ Ultrasound allows visualization of draining tracts and may reveal altered echogenicity of lymph nodes and other organs in small animals.¹⁰ In dogs with gastrointestinal disease, masses or obstructions may be detected with contrast imaging and ultrasound examination may reveal thickened segments of the gastrointestinal tract with concentric layers of altered echogenicity.⁷ A computed tomography (CT) scan was useful in delineating a retrobulbar mass in one cat.¹

Pathologic Findings

Pythiosis - Organisms can be visualized histologically.¹⁰ Pythium sp. does not stain well with hematoxylin and eosin staining, but it may be visualized as clear spaces with a band of eosinophilic material surrounding the border of the hyphal wall. Gridley- or Gomori’s methenamine silver- (GMS) stained preparations reveal infrequently septate hyphae ranging from 2.5-7.0 µm in diameter (Fig. 5). Hyphae have non-parallel walls and right angle branching.⁹

Figure 5. Histologic section of a mass associated with oomycete infection. The tissue contains hyphae with nonparallel walls, infrequent septa, and branching at right angles (GMS stain, image courtesy of Noah’s Arkive, University of Georgia).

Equine pythiosis - In horses with disseminated disease, multiple encapsulated nodular lesions with kunkers may be found in the internal organs, most notably the liver and lungs.^8,9,10 Effusion fluid often accumulates in the abdominal or thoracic cavity.⁸ When bone involvement is present, extensive periosteal reaction and osteolysis may be observed.⁹ Histologically, abundant microabscesses composed of eosinophils, lymphocytes, and macrophages are present.¹⁰ In chronic cases, eosinophilic granulomas with giant cells may be seen. Kunkers may be present in the center of many microabscesses.¹⁰

Canine and feline pythiosis - In dogs with gastrointestinal disease, laparotomy may reveal transmural thickening of segments of the gastrointestinal tract. Ulceration of the mucosa may occur anywhere along the alimentary tract and areas of necrosis may contain hyphae.³ Furthermore, diagnostic experience indicates that most cases of gastrointestinal pythiosis initially are mistaken for neoplasia. Characteristic tumor-like masses may be observed on affected organs in disseminated pythiosis.^6,7,10 Histologically, the lesions consist of eosinophilic granulomatous infiltrates composed of epithelioid macrophages, eosinophils, fibroblasts, neutrophils, multinucleate giant cells, and fibrous connective tissue have been observed.³

Pythiosis in cats usually involves the subcutis and it should be suspected when tumor-like masses are found as described above. Histologically, eosinophils, macrophages, and large lymphocytes are present within the lesions.¹⁰

Canine lagenidiosis - Postmortem examination of dogs with lagenidiosis has revealed more diffuse and frequent dissemination of infection. Reported findings include lymphadenitis, great vessel invasion, pulmonary lesions, ureteral obstruction, and invasion of the esophagus and trachea. Histologically, lagenidiosis is nearly identical to pythiosis. Internal structures of organisms are easier to see microscopically with lagenidiosis, and hyphae are frequently larger (7-25µm) than for Pythium sp.⁴ These organisms also are more apparent in hematoxylin and eosin-stained tissue sections than Pythium sp.

Diagnosis

The diagnosis of oomycete infections can be difficult due to the clinical and histological similarity between the oomycetes themselves as well as the similarities between oomycotic and zygomycotic infections.⁴ Some cases of lagiendiosis have probably been misdiagnosed as pythiosis. Though misdiagnosis of lagenidosis as pythiosis does not have clinical repercussions, misdiagnosis of zygomycosis as oomycosis may lead to inappropriate treatment regimens as zygomycetes are true fungi. Although there are histological differences between Lagenidium sp., Pythium insidiosum, and zygomycete organisms, they are not distinct enough for definitive diagnosis.⁴ A P. insidiosum-specific polyclonal antibody has been developed that does not stain Lagenidium sp. hyphae. The effectiveness of this primary antibody in immunohistochemical staining is currently under investigation.⁴

Currently, the diagnostic test of choice is culture of infected tissue followed by morphologic and molecular identification of the pathogen. Small pieces of tissue are placed on PYG agar and incubated at 37ºC. Growth should be apparent after 24 hours. Samples that must be shipped should arrive within 48 hours and should be packaged at ambient temperature (organisms may be impossible to culture if the tissue has been refrigerated or frozen). Bacterial contamination often prevents adequate growth of oomycetes. In such instances, antibiotic impregnated agar can prevent bacterial overgrowth.⁴ Even under the best of conditions, Pythium sp. is very difficult to grow and even harder to identify because it requires sporulation and identification of the zoospores.

PCR assays are available to detect oomycete DNA sequences in nucleic acid extracted from agar cultures, blood, or tissue biopsies. This technique also can differentiate zygomycosis, pythiosis, and lagenidiosis.⁴

Two ELISA tests are available for detection of anti-P. insidiosum antibodies in animals. Both of these tests are highly specific and sensitive.⁴ ELISA also may be useful in monitoring the response to treatment. Elimination of oomycete infection results in a serial decrease in antibody titer if treatment is successful.⁵

Treatment

The treatment of choice for oomycosis has been aggressive surgical resection.^3,10 Whenever possible, complete amputation of affected limbs may be curative.² In cases of advanced gastrointestinal and disseminated disease, total surgical excision may be impossible. In advanced gastrointestinal disease, infection usually involves the extensive portions of the alimentary tract and mesentery. Furthermore, the recurrence rate after surgery also is very high if the affected area is not completely excised.^3,10 Due to possible recurrence, it has been recommended that surgical excision be followed by systemic administration of antifungal drugs.¹⁰ However, antifungal therapy may not be effective because of the lack of ergosterol (the target molecule of antifungal drugs) in the plasma membranes of oomycetes. This probably explains why antifungal chemotherapy alone has shown very little success in treating oomycosis.⁵

Recently, immunotherapy using a newly formulated vaccine has been successful in treating cutaneous pythiosis in horses and dogs.⁵ This vaccine has been shown to be effective for both acute and chronic cutaneous pythiosis in the horse.^5,10 In one report, a dog was cured of cutaneous disease with only two injections of this vaccine given two weeks apart.⁵

For most oomycotic infections the prognosis is guarded to poor regardless of the advances in treatment. The reason is that many dogs have extensive gastrointestinal disease and many horses have multiple bone lesions at the time of initial presentation.^3,10 Disseminated disease and bone lesions are negative prognosticators in any species. The rate of recurrence after attempted surgical excision has also been a major factor in the failure of treatment.

References

1. Bissonnette KW, Sharp NJ, Dykstra MH, et al: Nasal and retrobulbar mass in a cat caused by Pythium insidiosum. J Med Vet Mycol 1991; 29:39-44.

2. Dykstra MJ, Sharp NJ, Olivry T, et al: A description of cutaneous-subcutaneous pythiosis in 15 dogs. Med Mycol 1999; 37:427-433.

3. Foil CS: Miscellaneous fungal infections. In: Greene CE (ed): Infectious Diseases of the Dog and Cat, 2nd ed. WB Saunders, Phildaelphia, 1998, pp.420-430.

4. Grooters AM, Hodgin C, Bauer RW, et al. Clinicopathological findings associated with Lagenidium sp. infection in 6 dogs: Initial description of an emerging oomycosis. J Vet Intern Med 2003; 17:637-646.

5. Hensel P, Greene CE, Medleau L, et al: Immunotherapy for treatment of multicentric cutaneous pythiosis in a dog. J Am Vet Med Assoc 2003; 223:215-218.

6. Jaeger GH, Rotstein DS, McHugh Law J: Prostatic Pythiosis in a dog. J Vet Intern Med 2002; 16:598-602.

7. Lijebjelke KA, Abramson C, Brockus C, Greene CE: Duodenal obstruction caused by infection with Pythium insidiosum in a 12-week old puppy. J Am Vet Med Assoc 2002; 220:1188-1191.

8. Reis Jl, Queiroz de Carvallo C, Nogueira RH, et al: Disseminated pythiosis in three horses. J Vet Microbiol 2003; 96:289-295.

9. Worster AA, Lillich JD, Cox JH, et al. Pythiosis with bone lesions in a pregnant mare. J Am Vet Med Assoc 2000; 216:1795-1798.

10. Mendoza L: Pythium insidiosum. http://medtech.cls.msu.edu/medtech/mendoza/

11. Image from http://plantclinic.cornell.edu/FactSheets/pythium/phythium_cycle_big.jpg

Acknowledgement

Image of the Horsehead Nebula can be found in the archive of NASA's Astronomy Picture of the Day, http://antwrp.gsfc.nasa.gov/apod/ap030713.html

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