Introduction

In 1984, the National Marine Fisheries Service (NMFS) began collecting undersized Hawaiian monk seal pups from the wild for rehabilitation on Oahu, Hawaii, to release back to the wild population. Twelve pups are currently in captivity at the Oahu holding facility, and nine have developed eye conditions consisting of severe bilateral corneal opacities, resulting in corneal edema, transient glaucoma, bullous keratitis, and transient conjunctivitis. This disease process has not previously been seen in wild or captive Hawaiian monk seals. Observers have not seen this problem in the wild. The etiology of this disease process is under investigation.

History

The twelve pups currently on Oahu were collected from French Frigate Shoals (FFS) in the Northwestern Hawaiian Islands, between 27 May and 31 July, 1995. At FFS, the seals were held for 2 to 21 days in a fenced shoreline pen. Three seals had conjunctivitis while in the holding pen at FFS.

In the nine affected pups, eye problems were reported 7 to 31 days after arrival to Oahu. Included were the three which also had reported eye problems at FFS. All nine pups reported to have conjunctivitis in the first few weeks on Oahu, went on to develop corneal edema, bullous keratitis, and transient conjunctivitis as well as eye pain 44 to 92 days following the resolution of initial eye problems. Of the three pups which did not develop corneal opacities, two had reports of eye problems but these symptoms were transient and did not involve the cornea or inflammation of the conjunctive. The corneal opacities and associated edema began centrally and progressed outward to the limbus. Conjunctivitis, photosensitivity, and occurred transiently after the entire cornea becomes opaque and a bullous keratitis developed. Vision is severely impaired for about 7-10 days at this stage of the disease. In 7 of the 9 affected seals, the right eye first demonstrated corneal opacity and edema; however, both eyes progressed similarly and to the same extent through the course of this disease process.

The order of occurrence of corneal opacities roughly corresponded to the seals' order and date of capture at FFS. Although the date of departure from FFS spans a 76 day period, all cases of corneal opacity occurred approximately 100 days after being held at FFS (93-119 days). The corneal opacities showed gross evidence of receding from the limbus in the first two affected seals at 49 and 53 days after detection. All affected pups are progressing toward resolution.

Diagnostic Efforts

At entry to the holding facility on Oahu, all 12 seals were evaluated clinically and blood samples were collected for complete blood counts (CBC), serum chemistries and, serology. Serology was done for toxoplasmosis, canine distemper, leptospirosis, Dirofilaria immitis, and canine parvovirus. Fecal samples were evaluated for intestinal parasites and Salmonella culture. None of these tests indicated disease.

During the initial period of conjunctivitis, after arrival on Oahu, the symptoms were limited to the eyes. Appetite, activity, CBC, serum chemistries remained essentially normal. CBC and serum chemistries were normal. Symptoms resolved in 3 - 4 days. About 80 days later when corneal opacities were observed an intense diagnostic effort began.

A botanist identified plants surrounding the holding pools and believed them not to be the cause for eye problems. Review of the paints, solvents and abrasives used previously on the pools found them not to be involved. The source water for these holding pools has the same source as is used for other pinnipeds (including four adult Hawaiian monk seals). Coliform counts are similar to previous years of pup rehabilitation. There has been no significant exposure to fresh water. The sunlight reflected from the walls and bottom of the pool is the same as in previous years. The diet of herring is from the same source as in previous years and is the same as is being fed to numerous other pinnipeds and cetaceans. Feral cats, mongoose, and mice from the vicinity of the holding pools have been necropsied. No ocular pathology was observed.

External ocular testing has been done. Conjunctival and corneal swabs have been evaluated by scanning electron microscopy and were unrevealing. Aerobic bacterial cultures have not identified a pathogen. Chlamydia culture/antigen testing and mycoplasma cultures have been done and are negative. Immediately prior to the bullous keratitis phase the intraocular pressures were elevated in two of the affected seals. Non anesthetized and restrained pups without corneal opacities had intraocular pressures of approximately 28 mmHg. Pups in the active bullous keratitis phase had intraocular pressures, when restrained, of approximately 56 mmHg. After the acute phase, intraocular pressures while restrained were similar or slightly less than those of unaffected pups. During the bullous keratitis phase the roughened cornea retained fluorescein stain.

Extensive serological evaluations have been done and some are still pending. Serum protein electrophoresis suggested immunocompetence but was otherwise unremarkable. Serum neutralizing antibody tests for canine distemper, canine parvovirus, canine corona virus, and infectious canine hepatitis were negative. Serological tests for leptospirosis, Dirofilaria immitis antigen, toxoplasmosis, and brucellosis were also negative. Serum neutralizing antibody tests for phocid calici virus and herpes simplex 11 were similar in affected as well as unaffected pups and to sera of pups from previous years. Serum vitamin A (sk) levels (30-33 mcg/dl) were similar for unaffected and affected pups. Serology for feline rhinotracheitis, feline calici virus, adenovirus panel, phocid distemper, and phocid herpes is still pending.

These pups were examined by eight veterinarians involved with marine mammals and several biologists. Their differential diagnostic list resulted in the previously stated testing. In addition five of the twelve pups were anesthetized with isoflurane and had complete ocular examinations by both a veterinary and human ophthalmologist. These examinations were documented by fundic, macro and still photography. Also there was video documentation of the anesthetic protocol and eye exams.

Tonometry found anesthetized intraocular pressures in unaffected and resolving eyes to be similar at approximately 12-15 mmHg. Slit lamp examination revealed corneal opacities and associated vascularization was limited to the anterior cornea) stroma. The corneal endothelium and immediately adjacent stroma was normal. In addition, the anterior eye chamber, iris, and lens were normal. Gonioscopy found no evidence of closed filtration angle, senechia, or filtration angle inflammation. Iris dilation was achieved with topical 10% neo Synephrine and 0.5 ml of 1/10,000 epinephrine. Fundic examination found a normal posterior chamber including macula, optic disc, tapetum, and retina. Aqueous humor (0.5 ml) was taken from each eye for further evaluation. Four 6 mm diameter, thin layer, corneal biopsies were taken. Three were preserved in 10% buffered formalin and one in glutaraldehyde. Five conjunctival biopsies were also taken. Four were preserved in 10% buffered formalin and one in glutaraldehyde. Anti Hawaiian monk seal antibody is being prepared for potential immuno histopathology. The formalin preserved biopsy samples are being investigated with light microscopy. No cells were found in the aqueous humor. Scanning electron microscopy on the aqueous humor is pending. Virus isolation of aqueous humor is still pending.

Treatments

At the Oahu holding facility during the initial conjunctivitis problems four of the nine affected pups were treated with topical triple antibiotic ointment for 3-5 days. Once the corneal opacities were observed oral Baytril (20 mg/kg ) and topical 1% chloramphenicol ointment were administered twice daily. Topical 1% chloramphenicol was discontinued and hyperosmotic sodium chloride ointment was substituted. After 5 days gentocin ophthalmic ointment was applied. After much consultation, oral (20 mg/kg twice daily) and topical 1% chloramphenicol were administered in conjunction with oral prednisone (0.50 mg/kg twice daily). One pup just beginning to display corneal opacities was given just oral prednisone (0.50 mg/kg twice daily). Subjectively none of these therapies altered the eventual progression of this disease process. The one pup treated with only oral prednisone has had a longer progression of the same pathological changes as those not similarly treated. Topical treatment with the nonsteroidal anti-inflammatory drug Profenal twice daily greatly reduced pain, photophobia, and see trauma to the eye. The use of Timoptic, lopidine, and Daranide did not grossly affect the progression of the corneal opacities.

Summary

While waiting for the serology, histopathology, electron microscopy, and virus isolation we continue to define the associations of different individuals at different periods of potential contagiousness. We have ruled out many etiologies in our investigation and many are still pending. We now have a well defined understanding of the pathology of this disease process as well as much more basic understanding of the anatomy of a Hawaiian monk seal eye. At this point in the investigation, all pathological changes are limited to the anterior cornea. Eye lesions on all affected pups are resolving. It is expected that all twelve pups will have vision compatible with survival in their natural habitat.

A definitive diagnosis and etiology are an extremely important element in the decision process for the release of these pups back to their native habitat. Along with our diagnostic efforts, decisions as to release criteria; evaluation of data in light of those criteria; and identification of qualified individuals who will participate is equally important.