Abstract

Case Report

On 14 September 1992, a 3.3-year-old 115-kg male spectacled bear (Tremarctos ornatus) presented with a 3-cm circular excoriation with alopecia on the fifth digit of both the right front and right hind feet. The bear had been observed licking the wounds which occasionally caused them to bleed. The exhibit consisted of an indoor cement floor with multiple stainless steel metal barred enclosures with guillotine shift doors. The outdoor exhibit consisted of gunnite, soil, vegetation, and a pool. The pool was drained and cleaned once per week. This bear was housed with his sibling and parents. The siblings and adult female were placed on exhibit together but kept separate from the adult male. The bear was started on 2880-mg trimethoprim sulfadiazine (Tribrissen, Coopers, Mundelein, IL, USA) SID for 10 days in a preferred food item. The wounds were sprayed with a dilute chlorhexidine solution (Nolvasan solution, Fort Dodge Laboratories, Inc., Fort Dodge, IA, USA).

Ten days later, the wounds were larger in diameter, moist and appeared more invasive. The bear was immobilized with tiletamine and zolazepam (Telazol, Fort Dodge Laboratories, Inc., Fort Dodge, IA, USA) 750 mg IM by dart. Physical examination revealed a 4-cm circular, moist, foul-smelling wound on the fifth digit of the front foot and a similar lesion on the right hind foot, which also had a missing toenail. Blood was collected from the femoral vein for a complete blood count (CBC), serum biochemical panel, and serum banking. Multiple skin biopsies were taken from each wound and submitted for histopathology and aerobic bacterial and fungal cultures. The animal recovered uneventfully. Histopathology revealed a deep fungal dermatitis with bacterial involvement. Aerobic bacterial culture revealed heavy growth of Escherichia coli, Proteus mirabilis, Klebsiella pneumoniae and an unidentified yeast. All the bacterial organisms were resistant to trimethoprim sulfadiazine but sensitive to enrofloxacin. All other diagnostic work was normal. Enrofloxacin (Baytril, Bayer Corporation, Shawnee Mission, KS, USA) 272 mg BID and fluconazole (Diflucan, Roerig, A Division of Pfizer Inc., NY, NY) 600 mg SID were started in a preferred food item and the trimethoprim sulfadiazine was discontinued. Daily spraying with dilute povidone-iodine solution (Vedadine solution, Vedco, Inc., St. Joseph, MO, USA) and topical ketoconazole cream (Janssen Pharmaceuticals, Mississauga, ON, Canada) was initiated. This regimen was continued for 6 weeks. On some days, the wounds seemed to be improving; however, if the bear licked the wounds, they looked worse. The wounds appeared to be very pruritic.

A second workup was performed 6 weeks after the first. The bear was anesthetized with tiletamine and zolazepam at the dosage used previously and maintained on isoflurane (AErrane, Ohmeda Pharmaceutical Products Division Inc., Liberty Corner, NJ, USA). Radiographs revealed an osteomyelitis of phalanx 3 (P3) on the fifth digit of both the right front and right hind feet. Deep pockets of purulent material were found on both wounds. Surgical amputation of P3 on both feet was performed. The exposed joint was flushed with amphotericin B prior to closing. Blood was drawn for CBC, serum biochemical panel, and blastomycosis serology. Tissue was submitted for fungal (two laboratories were used), mycobacterial, and aerobic and anaerobic bacterial cultures, and histopathology. The next day, the bear had licked out all the sutures from both amputation sites.

Histopathology revealed a multifocal nonsuppurative to granulomatous osteomyelitis with intralesional fungal hyphae. Aerobic bacterial culture revealed heavy growth of gram-positive and gram-negative bacteria, which were all sensitive to chloramphenicol and enrofloxacin. Neither fungal nor mycobacterial organisms were cultured. The bear was started on itraconazole (Sporanox, Janssen Pharmaceuticals, Mississauga, ON, Canada) 500 mg SID, PO and chloramphenicol 2000 mg BID, PO. The open incisions were sprayed with dilute chlorhexidine. The bear was housed on cement due to the open wounds.

Three weeks after the second workup, the bear presented with watery bloody diarrhea, vomiting and severe depression. Fecal examination for parasites was negative; however, numerous white blood cells were found on a Diff-Quik (Dade International Inc., Miami, FL, USA) fecal smear. Feces were submitted for enteric bacterial pathogen culture, and a piece of sloughed mucosa was submitted for histopathology. Itraconazole and chloramphenicol were discontinued, an electrolyte/glucose solution was placed in the bear’s water. The bear did not eat and would not take any medications orally for 3 days. The next day, the bear started eating small amounts of yogurt, sweet potato baby food, and primate chow. Clostridium perfringens was cultured from the feces. Histopathology on the mucosa submitted revealed an intestinal cast composed of mucus, necrotic debris with eosinophils and ingesta. A homogenous population of large gram-positive bacterial rods was also observed. The bear was started on 1000-mg metronidazole (250-mg tablets, USP, Sidmark Laboratories Inc., East Hanover, NJ, USA) BID, PO for 10 days. The wounds on the feet had improved at this point, presumably because the bear had not been licking them. The feces were back to normal after 5 days of metronidazole treatment.

Three weeks later, the right hind foot wound had a purulent discharge. The bear was immobilized with the same dose of tiletamine and zolazepam as previously used. The right front foot appeared to be almost completely healed. The right hind foot had an abscess that dissected between the lateral aspect of the third digit and involved most of the fourth digit. The wound was draining well. Radiographs of the right front foot were normal, while the right hind foot had a periosteal proliferation involving P1 and P2 of the fourth digit. Biopsy samples were taken for aerobic bacterial and fungal cultures and histopathology. Blood was taken for CBC, serum biochemical profile, and serum bank. The wounds were flushed with a dilute betadine solution. The animal was started back on itraconazole 500 mg SID orally. A lactobacillus product and yogurt were also given to the bear. Histopathology revealed a chronic purulent dermatitis/panniculitis with intralesional gram-positive cocci and a nodular eosinophilic dermatitis/panniculitis with intralesional fungal hyphae. Tissue sections were stained with an anti-Pythium antibody using an immunoperoxidase technique and were positive. At this point, the most likely organism producing the lesions was thought to be Pythium insidiosum. Fungal organisms were not isolated by culture. A diverse population of bacteria were cultured, as was the case previously. All other diagnostic work was normal.

Two weeks later, the nail on the fourth digit of the right hind foot was lost and the third digit continued to have a purulent discharge. The bear was immobilized using Telazol and isoflurane, as previously described. Both the third and fourth digits of the right hind foot were ulcerated and draining purulent material. The right front foot appeared to be healed but had a fluctuant area at the amputation site. The right popliteal lymph node was enlarged. Radiographs revealed an osteomyelitis of the third and fourth digits of the right hind foot; the right front foot appeared radiographically normal. Aspiration of the fluctuant area on the right front foot revealed purulent material. The fourth and fifth digits on the right hind foot were surgically removed. The distal metatarsal joints of both digits looked normal. The right front foot abscess was lanced and flushed. Blood was taken for CBC, a serum biochemical panel, and serum itraconazole levels (19 hours post administration). Tissue was submitted for fungal culture, aerobic and anaerobic bacterial culture, and histopathology. A biopsy of the enlarged lymph node was submitted for histopathology. Histopathology revealed a chronic eosinophilic inflammation with multiple to coalescing foci of degranulating eosinophils, collagen degeneration and intralesional hyphae. Itraconazole levels were 1.4 µg/ml (therapeutic range in other species 2–8 µg/ml). Bacterial culture produced heavy growth of Proteus vulgaris which was sensitive to enrofloxacin. Enrofloxacin was started at 250 mg BID, PO itraconazole, yogurt, and local flushing of the wound were continued.

Over the next 2 months, the wounds appeared to improve some days and looked worse on other days. A final immobilization, 7 months after the initial presentation, was performed to evaluate the wounds. The wounds had improved on the right hind foot, while the right front foot still had a fluctuant area that was filled with purulent material. Radiographs revealed progression of the osteomyelitis in both feet. Due to chronicity of the problem and the lack of response to treatment, the bear was euthanatized by IV barbiturate overdose (Beuthanasia-D Special, Schering-Plough Animal Health, Union, NJ, USA). Gross necropsy revealed an extremely enlarged right popliteal lymph node with purulent material noted on cut section. The right axillary lymph node was also enlarged but looked fairly normal on cut section. Histopathology revealed similar skin and bone changes as previously described. Lymphoid hyperplasia with sinusoidal eosinophilia was found on the enlarged lymph nodes. There was no evidence that the Pythium had disseminated to internal organs. A mold was reported to have been recovered and was forwarded to the Centers for Disease Control and Prevention for identification. Pythium insidiosum was cultured.

Medical problems were not noted in the other bears housed with this animal for a 5-year period. Recently, both parents of the bear in this case report developed obstructive lesions in the colon caused by Pythium insidiosum. Surgical resection of the affected gastrointestinal tract and anastomosis appear to have been curative in these two cases.

Discussion

Pythiosis is an invasive, eosinophilic to pyogranulomatous disease caused by Pythium insidiosum. It is an aquatic organism belonging in the order Peronosporales, phylum Oomycota, kingdom Protista. There are many different species of Pythium which are commonly found in soil and aquatic environments. They are considered important plant pathogens. Pythium insidiosum is the only member of this genus that causes disease in mammals. Pythiosis has been documented to cause disease in dogs, cats, horses, cattle and humans, although it is most common in dogs and horses. This is the first report of pythiosis in an ursid species. The disease occurs most commonly in tropical and subtropical areas worldwide. Most reports in the U.S. have originated from states bordering the Gulf of Mexico; however, there have been cases from Missouri, Georgia, North Carolina, South Carolina, and Tennessee. Pythium insidiosum is often found in stagnant water producing biflagellate zoospores that penetrate the animal through open skin. The environment of this spectacle bear was not excessively wet; however, there was a pool in the exhibit.

Three forms of the disease occur in domestic animal species: cutaneous disease, gastrointestinal disease, and rarely a systemic form. The cutaneous form is most commonly seen in the horse, although also occurs in other species. It most commonly affects the extremities, tailhead, perineum, and face. The lesions are initially an ulcerated plaque or erosion. These lesions enlarge rapidly into large masses with ulceration and draining tracts. Eventually, a hemorrhagic to purulent exudate is observed coming from the draining tract. The lesions noted in this bear followed a very similar course. There have been occasional reports of osteomyelitis developing in chronic cases of cutaneous pythiosis. Animals with cutaneous pythiosis are often extremely pruritic and may self-mutilate. The bear in this report seemed to continually aggravate his wounds by licking and rubbing.

The gastrointestinal form of pythiosis affects the stomach, proximal small intestine, distal small intestine, and colon most commonly; however, any area can potentially be involved. Vomiting, weight loss, and a palpable abdominal mass are common clinical findings.

This organism does cause disease in humans; however, the zoonotic potential is low. The motile zoospore is considered to be the major infective form of the organism. The zoospores do not form in tissue. There have been no reported cases of animal-to-animal, animal-to-human or human-to-animal transmission.

Histopathology and culture on biopsy specimens are the preferred method for confirmation of pythiosis. A nodular to pyogranulomatous dermatitis and panniculitis with foci of necrosis and large numbers of eosinophils are typical for tissue infected with P. insidiosum. The organism is difficult to see with routine hematoxylin-eosin (H&E), thus a Gomori methenamine silver stain is used. Hyphal structures are wide, occasionally septate, and irregularly branching and are often found in the center of granulomatous lesions. Gastrointestinal lesions are histologically similar to cutaneous lesions. The submucosa and muscularis layers are most severely affected. An immunoperoxidase assay was originally developed and validated on formalin-fixed tissues from horses with P. insidiosum. It has been used to confirm the diagnosis of pythiosis in dogs, horses, cats, and humans. It was helpful in developing a diagnosis sooner in this case. An immunofluorescence test is also available for detecting Pythium hyphal structures in cat, dog, and human tissue.

Pythium insidiosum grows readily on several media, including Sabouraud dextrose agar, brain-heart infusion medium, corn meal agar and vegetable extract agar. It is helpful to wash the sample in sterile saline with or without ampicillin to inhibit secondary bacterial growth. The organism usually grows within 24 hours.

Wide surgical excision of diseased tissue is the therapy of choice. When a limb is involved, amputation is recommended. Amputation was not possible in this case, because two limbs were involved. Histopathologic evaluation of arteries from the proximal end of amputation site and regional lymph nodes should be performed. Ascending arteritis is the most common form of the disease in humans. Prognosis is poor in all cases.

Pythium insidiosum is sensitive in vitro to miconazole, ketoconazole, and fluconazole, but not to amphotericin B. The azoles should not be effective in treating pythiosis, because this organism’s cell membrane does not contain ergosterol. Itraconazole has been used with limited success with the gastrointestinal form of the disease and has not been successful in treatment of the cutaneous form.

Immunotherapy using a vaccine has met with good success in horses with early lesions but has not been very successful in chronic cases (longer than 2 months).

The bloody diarrhea and vomiting in this case were most likely due to Clostridium perfringens enterotoxemia secondary to long-term use of antibiotic and antifungal drugs. This diagnosis was based on histopathology and culture results. A Clostridium perfringens enterotoxin assay was not performed.

Acknowledgments

We thank the animal husbandry and veterinary technical staffs at the Riverbanks Zoological Park for their care and technical assistance with the bear in this case report.