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Microsporidial Keratoconjunctivitis in a Pet Bearded Dragon (Pogona vitticeps)
CASE REPORT MICROSPORIDIAL KERATOCONJUNCTIVITIS IN A PET BEARDED DRAGON (POGONA VITTICEPS) Anna Martel-Arquette, DVM, Sue Chen, DVM, Dip. ABVP (Avian), Julie Hempstead, DVM, Dip. ACVO, Rebecca Pacheco, DVM, Natalie Antinoff, DVM, Dip. ABVP (Avian), and Leandro Teixeira, DVM, MSc, Dip. ACVP
Abstract A 4-month-old female bearded dragon (Pogona vitticeps) was presented with unilateral conjunctivitis. The conjunctivitis was not responsive to topical antibiotic or anti-inflammatory therapy and continued to progress in severity. Conjunctival tissue was sampled and microsporidial conjunctivitis was diagnosed on histopathological evaluation of submitted samples. Medical therapy was initiated and included topical itraconazole, systemic itraconazole, and systemic fenbendazole. Serial serum chemistry panels and complete blood counts were monitored for potential side effects associated with the medications. Despite treatment, the conjunctivitis progressed and enucleation was performed. Histopathology on the enucleated globe confirmed the diagnosis of microsporidial keratoconjunctivitis. Systemic itraconazole was administered at 5 mg/kg orally for approximately 3 months. No abnormalities were detected on hematologic diagnostic testing during the treatment period. A computed tomographic scan was performed to monitor for systemic lesions, but no abnormalities were detected. About 12 months after initial presentation, the bearded dragon is healthy and there are no overt signs of disease. Copyright 2017 Elsevier Inc. All rights reserved. Key words: microsporidiosis; conjunctivitis; keratoconjunctivitis; bearded dragon; itraconazole
A
4-month-old female bearded dragon (Pogona vitticeps) was presented with left superior blepharedema of unknown duration. The animal was obtained from a local pet store and the owners believed the animal had increased left blepharospasm at that time. The animal was maintained in a 40-gallon tank in which the environmental temperature within the enclosure varied from 27˚C (maintained by 1, 10.0 UVB lamp) to 36°C (maintained with 2, 100 W basking spot lamps). There were no other animals in the home. Calcium and vitamin D3 dusted crickets were fed on a daily basis. On examination, the left eye displayed evidence of blepharospasm with mucoid discharge. Ocular discharge cytology and culture were recommended but declined by the owner. The eye was cleansed with sterile ocular wash (Ocusoft, Inc., Richmond, TX USA) after administration of proparacaine (Akorn, Inc., Lake Forest, IL USA). Flurbiprofen (Bausch & Lomb Inc., Tampa, FL USA) was prescribed topically once daily for one week. When re-examined after 1 week of treatment, an anterior segment ophthalmic examination was performed with slit-lamp biomicroscopy (Kowa SL15, Kowas Co. Ltd, Torrance, CA USA), revealing
moderate blepharospasm, moderate blepharedema (inferior greater than superior), inferior chemosis, and hyperemia with overlying mucoid discharge lining the fornix and anterior
From the Department of Surgical Sciences, School of Veterinary Medicine, University of Wisconsin-Madison, Madison, WI, USA; the Gulf Coast Veterinary Specialists, Houston, TX, USA; and the Comparative Ocular Pathology Laboratory of Wisconsin (COPLOW), School of Veterinary Medicine. University of Wisconsin-Madison, Madison, WI, USA Address correspondence to: Anna Martel-Arquette, DVM, University of Wisconsin-Madison, 2015 Linden Dr., Madison, WI 53706. E-mail: [email protected]. Ó 2017 Elsevier Inc. All rights reserved. 1557-5063/17/2101-$30.00 http://dx.doi.org/10.1053/j.jepm.2017.07.006
Journal of Exotic Pet Medicine 26 (2017), pp 257–262
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fibrous tunic. The cornea was clear and no flare or other intraocular abnormalities were observed. Culture and antimicrobial sensitivity of bacteria isolated from a conjunctival swab was performed. Ofloxacin 0.3% (Akorn, Inc., Lake Forest, IL USA) was prescribed to be applied in the left eye every 12 hours pending culture and sensitivity results. Flurbiprofen 0.03% (Bausch & Lomb Incorporated, Tampa, FL USA) was increased in frequency to every 12 hours. The culture demonstrated abundant growth of a Salmonella spp. that was determined to be sensitive to ofloxacin. On re-examination 17 days postinitial evaluation, the owner noted progression of clinical signs despite treatment. On examination, the entire periorbita and both inferior and superior palpebrae were severely chemotic (Fig. 1). The left eyelids were held closed. To treat the inflammation, oral meloxicam 0.3 mg/kg orally, every 48 hours (Ceva Animal Health, LLC, Lenexa, KS USA) was prescribed. At that time, if medical management continued to fail, the authors would recommend surgical intervention to improve the patient’s level of comfort and informed the owner that there was a possibility that the bearded dragon may lose sight or use of the eye. Re-evaluation was performed 27 days after the initial presentation. The owner noted exacerbation of chemosis immediately after application of topical medications, which seemed to improve within a few hours. On follow-up 1 week later (4 weeks and 6 days after initial presentation), the animal continued to demonstrate severe clinical signs (e.g., periorbital edema and severe chemosis) with no improvement. The topical treatments (ofloxacin and flurbiprofen) were discontinued at that time. The oral meloxicam was continued and a recheck appointment was recommended in 7 to 10 days. If there was no improvement in the swelling, a conjunctival biopsy and subsequent histopathological assessment of the submitted tissue would be in order. Conjunctival incisional biopsy was performed under sedation 1 month and 23 days after initial presentation owing to the lack of treatment response of the eye. Hydromorphone 0.15 mg/kg intramuscular (West-ward, Eatontown, NJ USA) and alfaxalone 5 mg/kg intramuscular (Jurox, Inc., Kansas City, MO USA) were administered for the sedation. Using 0.3 mm Colibri forceps and iris scissors, a small slice of inferior bulbar conjunctiva was removed. The biopsy site was left to heal by second intention. Samples were submitted for aerobic culture and sensitivity and 2 5 8
histopathological evaluation. The bearded dragon was released from the hospital and prescribed ofloxacin drops to be placed in the affected eye every 8 hours postprocedure until the next recheck appointment. Meloxicam at 0.2 mg/kg was to be administered once daily by mouth postprocedure for 2 days. The culture and sensitivity of conjunctival tissue now demonstrated a light growth of Salmonella spp. that was sensitive to ofloxacin. The biopsy sample was sent to the Comparative Ocular Pathology Laboratory of Wisconsin at the University of Wisconsin-Madison for histopathological evaluation. Histopathology revealed a severe inflammatory infiltrate, composed largely of macrophages and fewer lymphocytes and plasma cells, infiltrating and expanding the conjunctival substantia propria with many intrahistiocytic, 3 to 5 μm diameter round to oval protozoal-like tachyzoites. The organisms were strongly positive under Gram stain, lightly Gomori silver stain positive, and negative under Fite’s stain. Based on these findings a final diagnosis of granulomatous conjunctivitis with intralesional protozoal-like organisms suggestive of a microsporidium (a primitive fungus) was reported (Fig. 2). At re-examination 10 days’ postbiopsy (2 months 2 days after initial presentation), fenbendazole 50 mg/kg orally every 24 hours for five days was started. On examination, the left eye appeared unchanged. Ofloxacin drops were continued every 8 hours in the left eye. Flurbiprofen therapy was again added to the therapeutic regimen, once daily in the left eye, in addition to oral meloxicam every 48 hours. Topical itraconazole 1% ophthalmic ointment (Compounded, Wedgewood Pharmacy,
FIGURE 1. Seventeen days postinitial presentation. Note increased chemosis of the inferior palpebra and obstruction of the cornea by edematous conjunctival tissue.
Martel-Arquette et al./Journal of Exotic Pet Medicine 26 (2017), pp 257–262
FIGURE 2. (A) Histopathology image of microsporidial organisms stained with Gram stain in the conjunctival tissue, and (B) same tissue stained with hematoxylin and eosin stain.
Swedesboro, NJ USA) was prescribed to be applied to left eye once daily. A complete blood count was performed and results were reported as within normal limits.1,2 A second re-examination 20 days postbiopsy (2 months 12 days postinitial presentation) demonstrated little change from the previous evaluation. Periorbital edema and moderate to marked chemosis continued to be present (Fig. 3). The complete blood count was repeated as previously described and the results were normal as per the previously reported reference range. The owner had not initiated the topical itraconazole treatment; therefore, this drug was again prescribed. Oral itraconazole was prescribed at 5 mg/kg orally, once daily for 30 days. The animal was re-examined 2 months and 29 days postinitial presentation (37 days postbiopsy) for assessment owing to acute onset of neurologic signs (fasiculations, “running around frantically”) at home. No neurologic abnormalities were noted. On examination, blepharospasm and moderate to
FIGURE 3. Three months postinitial presentation. Chemosis continues to progress and affects the whole periorbita (as compared to previous figure (Figure 1)). Note the continued obstruction of the cornea with the edematous conjunctival tissue.
marked chemosis (inferior palpebra greater than superior) was noted with overlying hyperemia. A thick crust was removed from the anterior fibrous tunic. Only a small window of clear cornea was visible when the conjunctival tissues were deflected towards the fornices. Topical itraconazole was increased to twice daily. Either subconjunctival/intralesional injection of an antifungal medication or enucleation was recommended. The owner elected to schedule an enucleation at that time. A standard transconjunctival enucleation was performed under general anesthesia 3 months and 11 days' postinitial presentation. The animal was sedated as previously described with hydromorphone 0.1 mg/kg intramuscularly and alfaxalone 15 alfaxalone 5 mg/kg intramuscularly, with Lactated Ringer’s 1.5 mL subcutaneously (Abbott Laboratories, North Chicago, IL USA) also being administered. Intubation was achieved with a modified 3.5 Fr red rubber tube. Anesthesia was maintained via isoflurane at 1.75% in a 1.5 L flow of oxygen. A lateral canthotomy (2 mm in length) was performed with Westcott tenotomy scissors. A conjunctival snip incision was made at 12:00 with Westcott tenotomy scissors, and the overlying Tenon’s capsule and conjunctiva dissected away from the sclera. A 360˚ incision was continued at the level of the sclera, 2 mm posterior to the limbus. All extraocular muscles were transected close to their globe attachment site. Steven’s tenotomy scissors were employed to cut the optic nerve and associated vessels. The orbit was packed with a small piece of gauze for hemostasis, and the third eyelid, eyelid margins (1 to 2 mm from gray line), and remaining conjunctiva excised. The orbit was flushed with sterile balanced salt solution. A splash block of 0.1 mL 2% lidocaine solution (2 mg) was provided prior to complete closure.1 The skin was apposed with 6-0 Nylon in 5
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interrupted cruciate sutures. The globe was placed in formalin and submitted to the Comparative Ocular Pathology Laboratory of Wisconsin at the University of Wisconsin-Madison for further histopathological evaluation. Hemostasis was performed with direct pressure with cotton-tipped applicator and hand held cautery (Bovie Medical, Clearwater, FL USA). Owing to blood loss during surgery, an additional 1.5 mL of Lactated Ringer’s was administered subcutaneously. Iron dextran 12.5 mg/kg intramuscular (MWI, Boise, ID USA) and vitamin B12 0.4 mg/kg subcutaneous (Sparhawk Laboratories, Inc., Lenexa, KS USA) were administered to support red blood cell regeneration. Ceftazidime 30 mg/kg intramuscular, every 72 hours for 5 doses was added to the treatment plan for the patient. Meloxicam 0.2 mg/kg orally every 24 hours was also prescribed for 7 days while the oral itraconazole was continued. At the re-examination day 10 postenucleation (3 months 20 days' postinitial presentation), the surgical incision was healing well. However, there were some small gray lesions noted rostral to the left nostril and craniodorsal to the left ear. Otherwise, the animal was doing well at home. The animal was maintained on the oral itraconazole treatment plan. Day 13 postenucleation (3 months 23 days’ postinitial presentation) the incision continued to heal well. The facial lesions noted at the previous visit were unchanged. A serum chemistry panel was performed at this time in order to assess liver enzymes (as a potential side effect of the itraconazole). Values were within reference ranges.1 After receiving the hematologic test results, itraconazole was to be continued for at least an additional 2 weeks. Suture removal was scheduled for 2 weeks later. Histology results indicated the patient had granulomatous keratoconjunctivitis, and numerous intralesional fungal organisms suggestive of Microsporidium sp. Suture removal was performed 25 days’ postenucleation (4 months and 4 days’ postinitial presentation). The owner was instructed to monitor the eye closely for any increase in edema and to return in 1 week to assess the patient’s weight. Itraconazole treatment was continued. About 1 month, 6 days’ postenucleation (4 months and 16 days after initial presentation) the animal continued to do well and gain weight at home. Owing to concerns about potential systemic spread of Microsporidium sp. Organisms from the eye, a computed tomographic (CT) scan 2 6 0
was recommended. Itraconazole by mouth was continued. Fenbendazole 50 mg/kg orally, every 24 hours once weekly for 3 treatments was prescribed in addition to the itraconazole. CT was performed 1 week later (4 months and 23 days after initial presentation). Using a GE Lightspeed QXi 4 CT unit, an axial noncontrast scan was performed at 120 kV and 160 mA. Slice acquisition thickness was 1.3 mm and slices were taken at 0.625 interleaving intervals. Alfaxalone was used for sedation at 15 mg/kg intramuscularly. Intubation was performed using a modified 5 Fr red rubber catheter, and the animal was ventilated with oxygen throughout the scan. No anesthetic complications occurred during the procedure. Imaging detected no sign recurrence of microsporidiosis or edema at the enucleation site or elsewhere in the skull. The surgical site continued to heal well with no complications. On re-examination 1 month 22 days’ postenucleation (5 months 2 days after initial presentation), the animal continued to thrive and gain weight. The surgical incision continued to heal well. A packed cell volume was taken to monitor for possible fenbendazole related anemia. The packed cell volume was 27%, which was normal. Oral itraconazole and fenbendazole treatments were continued. Medications were discontinued by the owner shortly after the last hospital visit and examination of the patient. An additional CT scan was recommended; however, this was declined by the owner. On follow-up for an unrelated condition 6 months and 11 days after initial presentation (3 months and 1 day postenucleation), the surgical
FIGURE 4. Approximately 3 months’ postenucleation. No further lesions noted on physical examination.
Martel-Arquette et al./Journal of Exotic Pet Medicine 26 (2017), pp 257–262
incision was completely healed (Fig. 4). The animal continued to thrive and grow. No signs of swelling were noted at the enucleation site. About 1-year postenucleation, the owner reported the patient continues to do well at home and does not appear to have any recurrence of granulomatous swelling in the area of the enucleated site. DISCUSSION Microsporidia are small, obligate intracellular, eukaryotic parasites that are characterized by the production of spores.3–8 Microsporidia belong to the phylum Microspora, which has been reclassified as fungi based on proteins found in the genome.4,8 These organisms are considered atypical fungi without mitochondria and have the smallest known genome.4,8 Microsporidial species are ubiquitous and highly resistant to environmental degradation.5,6,8,9 Microsporidia are reported to cause disease in a wide variety of invertebrate species and all classes of vertebrates.3–5,9–11 Most species infecting vertebrate hosts are found in fish, which has a large economic effect on industrial and recreational fisheries.8,11 Microsporidial spores can be identified by light or electronic microscopy on histopathologic samples. Spores are occasionally identified on Gram stains, modified trichrome stains, and Giemsa stains. Fluorescent stains may be used, but may provide a false positive.Polymerase chain reaction can also be used to diagnose microsporidiosis and DNA sequencing can be used to speciate the organism.8 Microsporidial infections are an increasing concern from a public health perspective.5–7 Zoonotic potential is of great concern; however, there has been no formal proof of zoonotic transmission.5,7,8,9,12 Microsporidia are able to adversely affect both immunocompetent and immunocompromised individuals, but are thought to be primarily opportunistic.6,7,10 Epidemiology, transmission routes, and sources of infection are not well understood at this time for microsporidian organisms.6–8 Though uncommon, it is well established that Microsporidium spp. can be the primary cause of keratitis.3–5,13 There is no established definitive treatment in humans for microsporidial keratoconjunctivitis.13 The treatment of choice for other forms of microspordiosis is albendazole, however there have been multiple reports of treatment failure for microsporidial
keratoconjunctivitis in both humans and birds.3–5,10 Albendazole is thought to cause selective degeneration of cytoplasmic microtubules in intestinal and tegmental cells of helminths. It is unclear how this drug is effective against microsporidial species. There are multiple reports of successful treatment with itraconazole, either as an initial therapeutic option or after albendazole failed to generate a treatment response for keratoconjunctivitis.4,5,11,13 Fumagillin, an antiangiogenesis agent, has also been suggested as an adjunctive method of topical treatment for microsporidial keratoconjunctivitis.3–5,10 However, this medication is not thought to penetrate deep corneal stroma and cannot be used systemically owing to its high toxic characteristics.4,10 In this case, the decision to treat with itraconazole was based on multiple studies in humans demonstrating treatment failure of microsporidial keratoconjunctivitis with albendazole and successful treatment with longterm systemic itraconazole.3,4,10,13 Though a study has been published suggesting that itraconazole may have toxic effects in bearded dragons, itraconazole has been used successfully with no adverse side effects for other fungal diseases by the authors.14,15 A serum chemistry panel was performed 6 weeks after starting systemic itraconazole to monitor for hepatotoxicity. In this case, no adverse effects were noted with systemic itraconazole. A combination of enucleation, fenbendazole, and itraconazole was successful at clearing microsporidial infection. No signs of systemic infection were noted on CT scan 6 weeks postenucleation. Additional samples were not available for speciation; which may have been helpful in determining a possible route of infection. The animal was purchased with left ocular blepharospasm. The owner also reported that the bearded dragon appeared lethargic and dehydrated at that time as well, which may have caused immunosuppression. No history of ocular trauma was reported. Microsporidial keratoconjunctivitis should be a differential diagnosis with any bearded dragon that presents with ocular lesions that are not responsive to antibiotic therapies. REFERENCES 1. Chow DW, Wong MY, Westermeyer HD: Comparison of two bupivicane delivery methods to control postoperative pain after enucleation in dogs. Vet Ophthalmol 5: 422-428, 2015
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2. Gibbons PM, Klaphake E, Carpenter JW: Reptiles, in Carpenter JW (ed): Exotic Animal Formulary. (ed 4). St. Louis, MO, Saunders/Elsevier, pp 137-138, 2013 3. Phalen DN, Logan KS, Snowden KF: Encephalitozoon hellem infection as the cause of a unilateral chronic keratoconjunctivitis in an umbrella cockatoo (Cacatua alba). Vet Ophthalmol 1:59-63, 2006 4. Font RL, Samaha AN, Keener MJ, et al: Corneal microsporidiosis: report of case, including electron microscopic observations. Ophthalmology 107(9):1769-1775, 2000 5. Canny CJ, Ward DA, Patton S, et al: Microsporidian keratoconjunctivitis in a double yellow-headed Amazon parrot (Amazona ochrocephala oratrix). J Avian Med Surg 12 (4):279-286, 1999 6. Lallo MA, Calábria P, Milanelo L: Encephalitozoon and Enterocytozoon (microsporidia) spores in stool from pigeons and exotic birds. Vet Parasitol 190:418-422, 2012 7. Haro M, Izquierdo F, Henriques-Gil N, et al: First detection and genotyping of human-associated microsporidia in pigeons from urban parks. Appl Environ Microbiol 71 (6):3153-3157, 2005 8. Vergneau-Grosset C, Larrat S: Microsporidiosis in vertebrate companion exotic animals. J Fungi 2(1):3-23, 2015
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9. Jacobson ER, Green DE, Undeen AH, et al: Systemic microsporidiosis in inland bearded dragons (Pogona vitticeps). J Zoo Wildl Med 29(3):315-323, 1998 10. Rossi P, Urbani C, Donelli G, et al: Resolution of microsporidial sinusitis and keratoconjunctivitis by itraconazole treatment. Am J Ophthalmol 127:210-212, 1999 11. Richter B, Graner I, Csokai J: Heterosporis anguillarum infection in a garter snake (Thamnophis sirtalis). J Comp Pathol 150:332-335, 2013 12. Lobo ML, Xiao L, Cama V, et al: Identification of potentially human-pathogenic Enterocytozoon bieneusi genotypes in various birds. Appl Environ Microbiol 72(11): 7380-7382, 2006 13. Sridhar MS, Sharma S: Microsporidial keratoconjunctivitis in a HIV-seronegative patient treated with debridement and oral itraconazole. Am J Opthalmol 136(4):745-746, 2003 14. Richter B, Csokai J, Graner I, et al: Encephalitozoonosis in two inland bearded dragons (Pogona vitticeps). J Comp Pathol 148:278-282, 2013 15. Van Waeyenberghe L, Baert K, Pasmans F, et al: Voriconazole, a safe alternative for treating infections caused by the Chrysosporium anamorph of Nannizziopsiss vriesii in bearded dragons (Pogona vitticeps). Med Mycol 48:880-885, 2010
Martel-Arquette et al./Journal of Exotic Pet Medicine 26 (2017), pp 257–262
Abstract A 4-month-old female bearded dragon (Pogona vitticeps) was presented with unilateral conjunctivitis. The conjunctivitis was not responsive to topical antibiotic or anti-inflammatory therapy and continued to progress in severity. Conjunctival tissue was sampled and microsporidial conjunctivitis was diagnosed on histopathological evaluation of submitted samples. Medical therapy was initiated and included topical itraconazole, systemic itraconazole, and systemic fenbendazole. Serial serum chemistry panels and complete blood counts were monitored for potential side effects associated with the medications. Despite treatment, the conjunctivitis progressed and enucleation was performed. Histopathology on the enucleated globe confirmed the diagnosis of microsporidial keratoconjunctivitis. Systemic itraconazole was administered at 5 mg/kg orally for approximately 3 months. No abnormalities were detected on hematologic diagnostic testing during the treatment period. A computed tomographic scan was performed to monitor for systemic lesions, but no abnormalities were detected. About 12 months after initial presentation, the bearded dragon is healthy and there are no overt signs of disease. Copyright 2017 Elsevier Inc. All rights reserved. Key words: microsporidiosis; conjunctivitis; keratoconjunctivitis; bearded dragon; itraconazole
A
4-month-old female bearded dragon (Pogona vitticeps) was presented with left superior blepharedema of unknown duration. The animal was obtained from a local pet store and the owners believed the animal had increased left blepharospasm at that time. The animal was maintained in a 40-gallon tank in which the environmental temperature within the enclosure varied from 27˚C (maintained by 1, 10.0 UVB lamp) to 36°C (maintained with 2, 100 W basking spot lamps). There were no other animals in the home. Calcium and vitamin D3 dusted crickets were fed on a daily basis. On examination, the left eye displayed evidence of blepharospasm with mucoid discharge. Ocular discharge cytology and culture were recommended but declined by the owner. The eye was cleansed with sterile ocular wash (Ocusoft, Inc., Richmond, TX USA) after administration of proparacaine (Akorn, Inc., Lake Forest, IL USA). Flurbiprofen (Bausch & Lomb Inc., Tampa, FL USA) was prescribed topically once daily for one week. When re-examined after 1 week of treatment, an anterior segment ophthalmic examination was performed with slit-lamp biomicroscopy (Kowa SL15, Kowas Co. Ltd, Torrance, CA USA), revealing
moderate blepharospasm, moderate blepharedema (inferior greater than superior), inferior chemosis, and hyperemia with overlying mucoid discharge lining the fornix and anterior
From the Department of Surgical Sciences, School of Veterinary Medicine, University of Wisconsin-Madison, Madison, WI, USA; the Gulf Coast Veterinary Specialists, Houston, TX, USA; and the Comparative Ocular Pathology Laboratory of Wisconsin (COPLOW), School of Veterinary Medicine. University of Wisconsin-Madison, Madison, WI, USA Address correspondence to: Anna Martel-Arquette, DVM, University of Wisconsin-Madison, 2015 Linden Dr., Madison, WI 53706. E-mail: [email protected]. Ó 2017 Elsevier Inc. All rights reserved. 1557-5063/17/2101-$30.00 http://dx.doi.org/10.1053/j.jepm.2017.07.006
Journal of Exotic Pet Medicine 26 (2017), pp 257–262
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fibrous tunic. The cornea was clear and no flare or other intraocular abnormalities were observed. Culture and antimicrobial sensitivity of bacteria isolated from a conjunctival swab was performed. Ofloxacin 0.3% (Akorn, Inc., Lake Forest, IL USA) was prescribed to be applied in the left eye every 12 hours pending culture and sensitivity results. Flurbiprofen 0.03% (Bausch & Lomb Incorporated, Tampa, FL USA) was increased in frequency to every 12 hours. The culture demonstrated abundant growth of a Salmonella spp. that was determined to be sensitive to ofloxacin. On re-examination 17 days postinitial evaluation, the owner noted progression of clinical signs despite treatment. On examination, the entire periorbita and both inferior and superior palpebrae were severely chemotic (Fig. 1). The left eyelids were held closed. To treat the inflammation, oral meloxicam 0.3 mg/kg orally, every 48 hours (Ceva Animal Health, LLC, Lenexa, KS USA) was prescribed. At that time, if medical management continued to fail, the authors would recommend surgical intervention to improve the patient’s level of comfort and informed the owner that there was a possibility that the bearded dragon may lose sight or use of the eye. Re-evaluation was performed 27 days after the initial presentation. The owner noted exacerbation of chemosis immediately after application of topical medications, which seemed to improve within a few hours. On follow-up 1 week later (4 weeks and 6 days after initial presentation), the animal continued to demonstrate severe clinical signs (e.g., periorbital edema and severe chemosis) with no improvement. The topical treatments (ofloxacin and flurbiprofen) were discontinued at that time. The oral meloxicam was continued and a recheck appointment was recommended in 7 to 10 days. If there was no improvement in the swelling, a conjunctival biopsy and subsequent histopathological assessment of the submitted tissue would be in order. Conjunctival incisional biopsy was performed under sedation 1 month and 23 days after initial presentation owing to the lack of treatment response of the eye. Hydromorphone 0.15 mg/kg intramuscular (West-ward, Eatontown, NJ USA) and alfaxalone 5 mg/kg intramuscular (Jurox, Inc., Kansas City, MO USA) were administered for the sedation. Using 0.3 mm Colibri forceps and iris scissors, a small slice of inferior bulbar conjunctiva was removed. The biopsy site was left to heal by second intention. Samples were submitted for aerobic culture and sensitivity and 2 5 8
histopathological evaluation. The bearded dragon was released from the hospital and prescribed ofloxacin drops to be placed in the affected eye every 8 hours postprocedure until the next recheck appointment. Meloxicam at 0.2 mg/kg was to be administered once daily by mouth postprocedure for 2 days. The culture and sensitivity of conjunctival tissue now demonstrated a light growth of Salmonella spp. that was sensitive to ofloxacin. The biopsy sample was sent to the Comparative Ocular Pathology Laboratory of Wisconsin at the University of Wisconsin-Madison for histopathological evaluation. Histopathology revealed a severe inflammatory infiltrate, composed largely of macrophages and fewer lymphocytes and plasma cells, infiltrating and expanding the conjunctival substantia propria with many intrahistiocytic, 3 to 5 μm diameter round to oval protozoal-like tachyzoites. The organisms were strongly positive under Gram stain, lightly Gomori silver stain positive, and negative under Fite’s stain. Based on these findings a final diagnosis of granulomatous conjunctivitis with intralesional protozoal-like organisms suggestive of a microsporidium (a primitive fungus) was reported (Fig. 2). At re-examination 10 days’ postbiopsy (2 months 2 days after initial presentation), fenbendazole 50 mg/kg orally every 24 hours for five days was started. On examination, the left eye appeared unchanged. Ofloxacin drops were continued every 8 hours in the left eye. Flurbiprofen therapy was again added to the therapeutic regimen, once daily in the left eye, in addition to oral meloxicam every 48 hours. Topical itraconazole 1% ophthalmic ointment (Compounded, Wedgewood Pharmacy,
FIGURE 1. Seventeen days postinitial presentation. Note increased chemosis of the inferior palpebra and obstruction of the cornea by edematous conjunctival tissue.
Martel-Arquette et al./Journal of Exotic Pet Medicine 26 (2017), pp 257–262
FIGURE 2. (A) Histopathology image of microsporidial organisms stained with Gram stain in the conjunctival tissue, and (B) same tissue stained with hematoxylin and eosin stain.
Swedesboro, NJ USA) was prescribed to be applied to left eye once daily. A complete blood count was performed and results were reported as within normal limits.1,2 A second re-examination 20 days postbiopsy (2 months 12 days postinitial presentation) demonstrated little change from the previous evaluation. Periorbital edema and moderate to marked chemosis continued to be present (Fig. 3). The complete blood count was repeated as previously described and the results were normal as per the previously reported reference range. The owner had not initiated the topical itraconazole treatment; therefore, this drug was again prescribed. Oral itraconazole was prescribed at 5 mg/kg orally, once daily for 30 days. The animal was re-examined 2 months and 29 days postinitial presentation (37 days postbiopsy) for assessment owing to acute onset of neurologic signs (fasiculations, “running around frantically”) at home. No neurologic abnormalities were noted. On examination, blepharospasm and moderate to
FIGURE 3. Three months postinitial presentation. Chemosis continues to progress and affects the whole periorbita (as compared to previous figure (Figure 1)). Note the continued obstruction of the cornea with the edematous conjunctival tissue.
marked chemosis (inferior palpebra greater than superior) was noted with overlying hyperemia. A thick crust was removed from the anterior fibrous tunic. Only a small window of clear cornea was visible when the conjunctival tissues were deflected towards the fornices. Topical itraconazole was increased to twice daily. Either subconjunctival/intralesional injection of an antifungal medication or enucleation was recommended. The owner elected to schedule an enucleation at that time. A standard transconjunctival enucleation was performed under general anesthesia 3 months and 11 days' postinitial presentation. The animal was sedated as previously described with hydromorphone 0.1 mg/kg intramuscularly and alfaxalone 15 alfaxalone 5 mg/kg intramuscularly, with Lactated Ringer’s 1.5 mL subcutaneously (Abbott Laboratories, North Chicago, IL USA) also being administered. Intubation was achieved with a modified 3.5 Fr red rubber tube. Anesthesia was maintained via isoflurane at 1.75% in a 1.5 L flow of oxygen. A lateral canthotomy (2 mm in length) was performed with Westcott tenotomy scissors. A conjunctival snip incision was made at 12:00 with Westcott tenotomy scissors, and the overlying Tenon’s capsule and conjunctiva dissected away from the sclera. A 360˚ incision was continued at the level of the sclera, 2 mm posterior to the limbus. All extraocular muscles were transected close to their globe attachment site. Steven’s tenotomy scissors were employed to cut the optic nerve and associated vessels. The orbit was packed with a small piece of gauze for hemostasis, and the third eyelid, eyelid margins (1 to 2 mm from gray line), and remaining conjunctiva excised. The orbit was flushed with sterile balanced salt solution. A splash block of 0.1 mL 2% lidocaine solution (2 mg) was provided prior to complete closure.1 The skin was apposed with 6-0 Nylon in 5
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interrupted cruciate sutures. The globe was placed in formalin and submitted to the Comparative Ocular Pathology Laboratory of Wisconsin at the University of Wisconsin-Madison for further histopathological evaluation. Hemostasis was performed with direct pressure with cotton-tipped applicator and hand held cautery (Bovie Medical, Clearwater, FL USA). Owing to blood loss during surgery, an additional 1.5 mL of Lactated Ringer’s was administered subcutaneously. Iron dextran 12.5 mg/kg intramuscular (MWI, Boise, ID USA) and vitamin B12 0.4 mg/kg subcutaneous (Sparhawk Laboratories, Inc., Lenexa, KS USA) were administered to support red blood cell regeneration. Ceftazidime 30 mg/kg intramuscular, every 72 hours for 5 doses was added to the treatment plan for the patient. Meloxicam 0.2 mg/kg orally every 24 hours was also prescribed for 7 days while the oral itraconazole was continued. At the re-examination day 10 postenucleation (3 months 20 days' postinitial presentation), the surgical incision was healing well. However, there were some small gray lesions noted rostral to the left nostril and craniodorsal to the left ear. Otherwise, the animal was doing well at home. The animal was maintained on the oral itraconazole treatment plan. Day 13 postenucleation (3 months 23 days’ postinitial presentation) the incision continued to heal well. The facial lesions noted at the previous visit were unchanged. A serum chemistry panel was performed at this time in order to assess liver enzymes (as a potential side effect of the itraconazole). Values were within reference ranges.1 After receiving the hematologic test results, itraconazole was to be continued for at least an additional 2 weeks. Suture removal was scheduled for 2 weeks later. Histology results indicated the patient had granulomatous keratoconjunctivitis, and numerous intralesional fungal organisms suggestive of Microsporidium sp. Suture removal was performed 25 days’ postenucleation (4 months and 4 days’ postinitial presentation). The owner was instructed to monitor the eye closely for any increase in edema and to return in 1 week to assess the patient’s weight. Itraconazole treatment was continued. About 1 month, 6 days’ postenucleation (4 months and 16 days after initial presentation) the animal continued to do well and gain weight at home. Owing to concerns about potential systemic spread of Microsporidium sp. Organisms from the eye, a computed tomographic (CT) scan 2 6 0
was recommended. Itraconazole by mouth was continued. Fenbendazole 50 mg/kg orally, every 24 hours once weekly for 3 treatments was prescribed in addition to the itraconazole. CT was performed 1 week later (4 months and 23 days after initial presentation). Using a GE Lightspeed QXi 4 CT unit, an axial noncontrast scan was performed at 120 kV and 160 mA. Slice acquisition thickness was 1.3 mm and slices were taken at 0.625 interleaving intervals. Alfaxalone was used for sedation at 15 mg/kg intramuscularly. Intubation was performed using a modified 5 Fr red rubber catheter, and the animal was ventilated with oxygen throughout the scan. No anesthetic complications occurred during the procedure. Imaging detected no sign recurrence of microsporidiosis or edema at the enucleation site or elsewhere in the skull. The surgical site continued to heal well with no complications. On re-examination 1 month 22 days’ postenucleation (5 months 2 days after initial presentation), the animal continued to thrive and gain weight. The surgical incision continued to heal well. A packed cell volume was taken to monitor for possible fenbendazole related anemia. The packed cell volume was 27%, which was normal. Oral itraconazole and fenbendazole treatments were continued. Medications were discontinued by the owner shortly after the last hospital visit and examination of the patient. An additional CT scan was recommended; however, this was declined by the owner. On follow-up for an unrelated condition 6 months and 11 days after initial presentation (3 months and 1 day postenucleation), the surgical
FIGURE 4. Approximately 3 months’ postenucleation. No further lesions noted on physical examination.
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incision was completely healed (Fig. 4). The animal continued to thrive and grow. No signs of swelling were noted at the enucleation site. About 1-year postenucleation, the owner reported the patient continues to do well at home and does not appear to have any recurrence of granulomatous swelling in the area of the enucleated site. DISCUSSION Microsporidia are small, obligate intracellular, eukaryotic parasites that are characterized by the production of spores.3–8 Microsporidia belong to the phylum Microspora, which has been reclassified as fungi based on proteins found in the genome.4,8 These organisms are considered atypical fungi without mitochondria and have the smallest known genome.4,8 Microsporidial species are ubiquitous and highly resistant to environmental degradation.5,6,8,9 Microsporidia are reported to cause disease in a wide variety of invertebrate species and all classes of vertebrates.3–5,9–11 Most species infecting vertebrate hosts are found in fish, which has a large economic effect on industrial and recreational fisheries.8,11 Microsporidial spores can be identified by light or electronic microscopy on histopathologic samples. Spores are occasionally identified on Gram stains, modified trichrome stains, and Giemsa stains. Fluorescent stains may be used, but may provide a false positive.Polymerase chain reaction can also be used to diagnose microsporidiosis and DNA sequencing can be used to speciate the organism.8 Microsporidial infections are an increasing concern from a public health perspective.5–7 Zoonotic potential is of great concern; however, there has been no formal proof of zoonotic transmission.5,7,8,9,12 Microsporidia are able to adversely affect both immunocompetent and immunocompromised individuals, but are thought to be primarily opportunistic.6,7,10 Epidemiology, transmission routes, and sources of infection are not well understood at this time for microsporidian organisms.6–8 Though uncommon, it is well established that Microsporidium spp. can be the primary cause of keratitis.3–5,13 There is no established definitive treatment in humans for microsporidial keratoconjunctivitis.13 The treatment of choice for other forms of microspordiosis is albendazole, however there have been multiple reports of treatment failure for microsporidial
keratoconjunctivitis in both humans and birds.3–5,10 Albendazole is thought to cause selective degeneration of cytoplasmic microtubules in intestinal and tegmental cells of helminths. It is unclear how this drug is effective against microsporidial species. There are multiple reports of successful treatment with itraconazole, either as an initial therapeutic option or after albendazole failed to generate a treatment response for keratoconjunctivitis.4,5,11,13 Fumagillin, an antiangiogenesis agent, has also been suggested as an adjunctive method of topical treatment for microsporidial keratoconjunctivitis.3–5,10 However, this medication is not thought to penetrate deep corneal stroma and cannot be used systemically owing to its high toxic characteristics.4,10 In this case, the decision to treat with itraconazole was based on multiple studies in humans demonstrating treatment failure of microsporidial keratoconjunctivitis with albendazole and successful treatment with longterm systemic itraconazole.3,4,10,13 Though a study has been published suggesting that itraconazole may have toxic effects in bearded dragons, itraconazole has been used successfully with no adverse side effects for other fungal diseases by the authors.14,15 A serum chemistry panel was performed 6 weeks after starting systemic itraconazole to monitor for hepatotoxicity. In this case, no adverse effects were noted with systemic itraconazole. A combination of enucleation, fenbendazole, and itraconazole was successful at clearing microsporidial infection. No signs of systemic infection were noted on CT scan 6 weeks postenucleation. Additional samples were not available for speciation; which may have been helpful in determining a possible route of infection. The animal was purchased with left ocular blepharospasm. The owner also reported that the bearded dragon appeared lethargic and dehydrated at that time as well, which may have caused immunosuppression. No history of ocular trauma was reported. Microsporidial keratoconjunctivitis should be a differential diagnosis with any bearded dragon that presents with ocular lesions that are not responsive to antibiotic therapies. REFERENCES 1. Chow DW, Wong MY, Westermeyer HD: Comparison of two bupivicane delivery methods to control postoperative pain after enucleation in dogs. Vet Ophthalmol 5: 422-428, 2015
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2. Gibbons PM, Klaphake E, Carpenter JW: Reptiles, in Carpenter JW (ed): Exotic Animal Formulary. (ed 4). St. Louis, MO, Saunders/Elsevier, pp 137-138, 2013 3. Phalen DN, Logan KS, Snowden KF: Encephalitozoon hellem infection as the cause of a unilateral chronic keratoconjunctivitis in an umbrella cockatoo (Cacatua alba). Vet Ophthalmol 1:59-63, 2006 4. Font RL, Samaha AN, Keener MJ, et al: Corneal microsporidiosis: report of case, including electron microscopic observations. Ophthalmology 107(9):1769-1775, 2000 5. Canny CJ, Ward DA, Patton S, et al: Microsporidian keratoconjunctivitis in a double yellow-headed Amazon parrot (Amazona ochrocephala oratrix). J Avian Med Surg 12 (4):279-286, 1999 6. Lallo MA, Calábria P, Milanelo L: Encephalitozoon and Enterocytozoon (microsporidia) spores in stool from pigeons and exotic birds. Vet Parasitol 190:418-422, 2012 7. Haro M, Izquierdo F, Henriques-Gil N, et al: First detection and genotyping of human-associated microsporidia in pigeons from urban parks. Appl Environ Microbiol 71 (6):3153-3157, 2005 8. Vergneau-Grosset C, Larrat S: Microsporidiosis in vertebrate companion exotic animals. J Fungi 2(1):3-23, 2015
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9. Jacobson ER, Green DE, Undeen AH, et al: Systemic microsporidiosis in inland bearded dragons (Pogona vitticeps). J Zoo Wildl Med 29(3):315-323, 1998 10. Rossi P, Urbani C, Donelli G, et al: Resolution of microsporidial sinusitis and keratoconjunctivitis by itraconazole treatment. Am J Ophthalmol 127:210-212, 1999 11. Richter B, Graner I, Csokai J: Heterosporis anguillarum infection in a garter snake (Thamnophis sirtalis). J Comp Pathol 150:332-335, 2013 12. Lobo ML, Xiao L, Cama V, et al: Identification of potentially human-pathogenic Enterocytozoon bieneusi genotypes in various birds. Appl Environ Microbiol 72(11): 7380-7382, 2006 13. Sridhar MS, Sharma S: Microsporidial keratoconjunctivitis in a HIV-seronegative patient treated with debridement and oral itraconazole. Am J Opthalmol 136(4):745-746, 2003 14. Richter B, Csokai J, Graner I, et al: Encephalitozoonosis in two inland bearded dragons (Pogona vitticeps). J Comp Pathol 148:278-282, 2013 15. Van Waeyenberghe L, Baert K, Pasmans F, et al: Voriconazole, a safe alternative for treating infections caused by the Chrysosporium anamorph of Nannizziopsiss vriesii in bearded dragons (Pogona vitticeps). Med Mycol 48:880-885, 2010
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