Abstract
A severe poxvirus infection occurred in three pudu (Pudu puda), resulting in two fatalities. Cutaneous ulcers with mucopurulent exudate were present on periorbital skin, nose, lip margins, coronary bands, and teats, and mucosal ulcers were present in the oral cavity, esophagus, and forestomach. In the two fatalities, a secondary systemic fungal infection also occurred. Affected animals were leukopenic, hypocalcemic, hyperphosphatemic, and had elevated serum alkaline phosphatase, alanine aminotransferase, and aspartate aminotransferase levels. One case was treated with cidofovir, 5 mg/kg IV every 7 days for four treatments. Complete recovery occurred in the treated animal in 4 weeks. This is the second report of poxvirus infection in pudu, and the first report describing clinical presentation, presence of secondary fungal infection, and successful treatment.
Introduction
Poxvirus infections in ungulates have been described in a variety of domestic and nondomestic ungulates. A novel poxvirus infection has been described in pudu (Pudu puda) in captivity.9,10 This report details the second outbreak of this virus in a zoological collection, complicated with a significant systemic fungal infection. The report describes clinical presentation and antiviral therapy with cidofovir.
Case Reports
The affected pudu group consisted of an adult pair and a male calf. At 1 month of age, the calf was examined for a suspected abrasion with purulent exudate on the left side of the muzzle near the preorbital scent gland. Treatment consisted of topical antibiotic cream and systemic antibiotics. Results of complete blood count (CBC) and serum biochemical profile were within normal limits for the species.6 Examination 1 week later revealed mild hyperthermia (39.6°C) and persistence and extension of the cutaneous lesions, now appearing as ulcers with adherent purulent debris. A small ulcer was also present on the tongue tip, and leukopenia (WBC=1600 cells/µl), hypocalcemia (7 mg/dl), hypoproteinemia (4.4 g/dl), and elevated aspartate aminotransferase (AST) (387 IU/L) were present. A presumptive diagnosis of poxvirus infection was reached based on lesions and progression consistent with the previous report.9,10 Supportive care (fluid and antibiotic therapy and nutritional support) was continued for 1 week. At that time, ulcers were present at the mucocutaneous margins of the lips and nose, anterior to the preorbital scent glands, and bilaterally on the lateral aspects of the forelimbs near the coronary band. Ulcers had adherent purulent exudate forming crusts. Extensive ulceration was also present on the tongue and at the dorsal oral cavity. The calf was depressed, inappetent, and febrile. Increased respiratory sounds were auscultable in all lung fields, and dyspnea was evident. Results of CBC confirmed the persistence of leukopenia (WBC=3400 cells/µl) and sickle cells, and serum biochemical profile confirmed hypocalcemia (6.8 mg/dl), hypoproteinemia (5.9 g/dl), and elevated AST (3171 IU/L), and indicated hypoglycemia (37 mg/dl), hyperphosphatemia (12.0 mg/dl), and elevated alanine aminotransferase (353 IU/L). Due to continued deterioration and grave prognosis, the animal was euthanatized (14 days after initial presentation).
The second case involved the adult female, the dam of case 1. First clinical signs were noted 2 days after the appearance of lesions in the calf. Initial cutaneous lesions were present bilaterally on the rear limbs from the coronary bands to the tarsometatarsal joint, and bilaterally on the forelimbs at the lateral aspect of the carpus. Results of CBC and serum biochemical profile were within normal limits, and therapy consisted of topical and systemic antibiotics as with the first case. One week after initial presentation, areas of dermatitis on the extremities were significantly worse, and swelling had developed ventral to the right orbit. Marked respiratory stridor was present.
Repeated CBC remained within normal limits, and serum biochemical profile revealed hypocalcemia (7.9 mg/dl), hyperphosphatemia (11.6 mg/dl), and elevated AST (1376 IU/L). Two days later, extensive lingual and oral cavity ulceration was noted. In spite of supportive care, the animal’s condition continued to decline. A deep corneal ulcer developed in the left eye, and ulcers were present at the mucocutaneous junctions of the eyelids and oral cavity, planum nasale, distal limbs, and ventral abdomen over the mammary glands. Results of CBC and serum profile taken 2 days later revealed leukopenia (WBC=3,400 cells/µl), continued hypocalcemia, hyperphosphatemia, and elevated AST, and the development of hypoglycemia, hypoproteinemia, and elevated ALT, and high creatine phosphatase (CK) (4768 IU/L). The pudu died overnight, 12 days after initial presentation.
Postmortem examinations were performed on the pudus. In both cases, multiple ulcers were present at the tongue tip and hard palate, and on incision of the salivary gland and mandibular lymph nodes a purulent exudate was expressed. Lungs had depressed, dark red areas with scattered firmer 3–5-mm foci which were dark red or tan on section. Prescapular and mandibular lymph nodes were enlarged three to five times normal size. Scattered high numbers of ring-like ulcers up to 3-mm diameter were present in the forestomach of the infant. Histologically, tissue from the lip had multifocal epithelial necrosis and areas of hydropic swelling. In the esophagus, there was focally extensive thickening of the mucosa with eosinophilic intracytoplasmic inclusion bodies and small areas of full-thickness necrosis. Similar lesions were present in the reticulum of the infant. The lungs had discrete areas of lytic necrosis centered on arteries. Multiple dense mats of fungal hyphae were associated with these in the infant. Lymph nodes from both necropsy cases had fungal hyphae and rare bacterial colonies on a background of necrotic lymphoid tissue. Fungal hyphae were noted in pyogranulomatous skin lesions on the infant, with vascular invasion. A diagnosis of disseminated poxvirus infection with secondary fungal infection was made. Poxvirus infection was confirmed via electron microscopy and serologic testing.
Case 3 involved the adult male, which did not show clinical signs at the time the adult female and calf became ill. When the first two pudus began to show clinical signs, the male was moved into isolation. Mild facial swelling was noted 10 days after the female pudu began showing signs; however, no cutaneous pox lesions were noted until the day after the adult female died. At this point, the pudu was examined due to right ocular discharge. Sloughing skin with underlying seropurulent discharge was present caudal to the right eye, over the dorsal nose, and at the coronary bands of both tarsi and the right front foot. The lesions were debrided and cleaned with topical disinfectant. The following day, the animal was depressed, febrile (40.6°C), and dyspneic. Results of CBC indicated leukopenia, and serum biochemical profile indicated hypocalcemia, hyperphosphatemia, elevated AST, and high CK.
Based on the previous cases, the prognosis for survival was considered poor. Antiviral therapy with cidofovir (Vistide, Gilead Sciences, Foster City, CA, USA) 5 mg/kg IV every 7 days for four treatments was initiated. Intravenous fluid therapy and oral probenecid (Benemid, Merck and Co., Inc., West Point, PA, USA) was given concurrently with each treatment in the following regimen. An indwelling catheter was placed in a cephalic vein, and 100 ml normal saline was administered IV and 250 mg probenecid was given orally. One hour later, cidofovir 5 mg/kg diluted in 30 ml normal saline was given IV. One hour later, 100 ml normal saline was given IV, 250 mg probenecid was given PO, and the catheter was removed. Within 2 days of the initial treatment, the animal appeared less depressed. Superficial lesions were notably less exudative, and facial swelling seemed diminished. Antibiotic delivery was changed to enrofloxacin (Baytril, Bayer Corp., Shawnee Mission, KS, USA) 2.5 mg/kg PO, SID. At this point, necropsy results from the first two cases were available and indicated secondary fungal disease was an important factor. Itraconazole (Sporanox, Janssen Pharmaceutical, Inc., Titusville, NJ, USA) 100 mg PO, SID for 14 days was initiated.
At 1 week after initiation of antiviral therapy, cutaneous lesions were significantly improved. No new lesions had developed, and existing lesions became nonexudative and healed. Repeated examination at weekly intervals confirmed that no new cutaneous or mucocutaneous lesions had developed.
Discussion
Poxvirus infection in pudus has been reported, the virus described, and serologic cross-reactivity evaluated. Based on ultrastructural features, size, and lack of neutralization by antisera to multiple known poxviruses, the investigators suggested that the isolate was a novel poxvirus.9,10 That case description indicated illness in eight of nine pudus, with one death.9,10 Gross lesions were limited to periorbital area and/or oral cavity in the surviving animals, and also on the palate, esophagus, rumen, and reticulum of the single fatal case. As with the current case, the animals were housed in a single species exhibit, and no recent additions had occurred.
The clinical presentation of poxvirus infection in pudu includes cutaneous and mucocutaneous ulcers and vesicles, as with other poxvirus infections. In this outbreak, ulcers at the coronary bands, cornea, and mammae occurred, which were not reported in the previous outbreak.10 In addition, significant pneumonia was present in the two cases that died. These animals were consistently febrile and dyspneic, with coarse lung sounds. The presence of disseminated fungal disease was suspected to be due to immunosuppression secondary to viremia. In the two fatal cases, the progression from first evidence of cutaneous lesions to death was approximately 2 weeks.
Clinical pathology findings in these cases were consistent, with early development leukopenia typical for viral infection. Electrolyte abnormalities (hypocalcemia, hyperphosphatemia) and high enzyme levels (AST, ALT) may be due to extensive tissue damage that results from the necrosis associated with poxvirus lesions. As cases progress, hypoglycemia and hypoproteinemia develop, probably due to decreased nutritional intake. It is presumed that the high CK values were secondary to repeated IM injections during treatment attempts. In the case that recovered, all CBC and serum biochemical profile abnormalities resolved coincident with clinical evidence of improvement.
The source of the virus in this outbreak could not be determined. As with the previously reported case, no recent exposure to new conspecifics or other species occurred. It is possible that a prolonged carrier state exists, and stress (such as the parturition in the recent case) initiated a recurrence. It is also possible that the poxvirus is latent in another species and manifests itself as a more serious disease in the aberrant host. Pudus are South American Cervidae, and at our institution are housed in a barn that is predominantly African antelope (Bovidae). However, no poxvirus disease has been documented or suspected in the remaining collection.
Antiviral drug therapy is not routinely used for animal poxviruses. Cidofovir is a nucleoside analog of deoxycytidine monophosphate that has shown activity against herpesviruses, as well as other virus infections, including poxvirus.1,7 This drug is licensed for treatment of cytomegalovirus (CMV) retinitis in human patients with human immunodeficiency virus infection.3 Cidofovir suppresses viral replication by selective inhibition of viral DNA synthesis.3 While not approved for other viral infections, experimental research has suggested that cidofovir is effective against poxvirus infection.1,2,4,6,7 An anecdotal report of three human patients with HIV being treated with cidofovir for CMV retinitis indicates dramatic improvement of concurrent molluscum contagiosum virus dermatitis.7 No reports of the use of cidofovir against animal poxvirus infections were found. Dose-dependent nephrotoxicity is the major adverse reaction of cidofovir.3 To control nephrotoxicity, patient pretreatment evaluation is recommended, along with IV normal saline hydration. In addition, the concurrent administration of probenecid is recommended. Probenecid is a renal tubular transport-blocking agent.8 Administration prolongs the half-life of cidofovir, resulting in a prolonged period between doses.
The treatment regimen provided concomitant hydration and probenecid therapy, an appropriate dose, and a manageable treatment frequency. Patient monitoring did not reveal any evidence of renal impairment or leukopenia, the most common side effects in humans. Although only a single case is represented, the resolution of lesions, clinical signs, and clinical pathology abnormalities suggest that the treatment was both successful and safe. Attention to possible secondary infection is necessary. The presence of systemic fungal disease in these cases may suggest an immunocompromised state secondary to the viral infection.
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