Experimental Ocular Onchocerciasis in Cynomolgus Monkeys

IV. Chorioreriniris Elicited by Onchocerca volvulus Microfilariae

Richard D. Semba,* John J. Donnelly,t Elaine Young,* W. Richard Green,* Alan L. Scorr,:j: and Hugh W. Taylor*

Onchocerciasis is a major cause of blindness worldwide, and much of the blindness is caused by onchocercal chorioretinitis. In an experimental animal model for ocular onchocerciasis, intravitreal injections of 10,000 live Onchocerca volvulus microfilariae isolated from infected humans into the eyes of cynomolgus monkeys (Macaca fascicularis) resulted in patchy, progressive loss of retinal pigment with pigment clumping. Areas of pigment loss were less extensive in animals that had been sensitized with microfilariae. Intravitreal injections of dead O. volvulus microfilariae resulted in mild vitritis with relatively less clinical change noted in the retina and choroid. Histopathologic examination revealed thinning and loss of outer retinal layers with pigment migration into the retina, and inflammation was more pronounced in eyes that received live microfilariae. Clinical changes appeared in eyes receiving live microfilariae before the development of significant antibody or cell-mediated immune responses. O. volvulus microfilariae appear to be more suitable than O. lienalis microfilariae in producing lesions which resemble human onchocerciasis in the primate model. Invest Ophthalmol Vis Sci 32:1499- 1507,1991

Onchocerciasis, a filarial infection caused by On- phils were produced by intravitreal injections of up to chocerca volvulus, affects more than 18 million people 500 live O. lienalis microfilariae. However, O. liena- worldwide1 and is a leading cause of blindness. A lis, a related species to O. volvulus, is the species which characteristic chorioretinitis and chorioretinal scar- infects cattle. Ocular inflammation elicited by O. lien- ring account for nearly one half of this blindness. The alis microfilariae in the experimental primate model pathogenesis of the fundus lesion is unclear in part was much greater than that seen in human ocular on- due to the scarcity of affected human eyes for patho- chocerciasis. We tried to determine whether O. volvu- logic studies and the lack of detailed longitudinal oph- lus microfilariae isolated from infected humans thalmologic study of affected patients. would elicit onchocercal chorioretinitis in the eyes of An experimental animal model using intravitreal cynomolgus monkeys and whether this would more injections of O. lienalis microfilariae in cynomolgus closely resemble human ocular onchocerciasis both monkeys recently facilitated the study of the immuno- clinically and histopathologically. Both live and dead pathology and pathogenesis of onchocercal chorioreti- O. volvulus microfilariae were used in intravitreal in- nitis.2"4 Chorioretinal changes, including retinal pig- jections to determine whether direct invasion of the ment epithelial hypertrophy, hyperplasia, and loss of retina and choroid was necessary to cause chorioreti- pigment, and choroiditis involving primarily eosino- nal changes resembling human ocular onchocerciasis. Humoral and cell-mediated immune responses to on- From the *Dana Center for Preventive Ophthalmology, The chocercal antigens were studied to help elucidate the Wilmer Institute and the School of Hygiene and Public Health, The role of immune mechanisms in the pathogenesis of Johns Hopkins University, Baltimore, Maryland, the fScheie Eye onchocercal chorioretinitis. Institute, University of Pennsylvania School of Medicine, Philadel- phia, Pennsylvania, and the ^Department of Immunology and In- fectious Diseases, The School of Hygiene and Public Health, The Materials and Methods Johns Hopkins University, Baltimore, Maryland. Supported in part by NIH grants K11 EY00286 (RDS), EY01765 Skin biopsy specimens were obtained from humans (EY), EY03984 (JJD), EY06616 (JJD), and by grants from the with onchocerciasis who were undergoing therapeutic World Health Organization Special Programme for Research and nodulectomy on the Liberian Agricultural Company Training in Tropical Diseases (RDS) and Onchocerciasis Control Programme Chemotherapy Project (JJD). rubber plantation, Grand Bassa County, Liberia. All Reprint requests: Richard Semba, MD, The Wilmer Eye Insti- individuals were characterized by onchocercal nod- tute, The Johns Hopkins Hospital, Baltimore, MD 21205. ules (>5) and high densities of microfilariae in the

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skin (>70 microfilariae/mg skin). Specimens contain- centesis was done after the intravitreal injection to ing microfilariae were cryopreserved in 6% dimethyl relieve intraocular pressure. sulfoxide in RPMI-1640 media (Gibco, Grand Island, Slit-lamp examination, anterior segment photogra- NY) and stored in liquid nitrogen. To prepare micro- phy, direct and indirect ophthalmoscopy, fundus pho- filariae for injection, the specimens were thawed at tography, and fliiorescein angiography were done at 37°C, washed with fresh RPMI-1640 medium con- baseline and at 1,3, 10, 18, 35, 41, and 53 days after taining 100 U/ml of penicillin, 100 Mg/ml of strepto- intravitreal injection. Inflammation of the anterior mycin, and 10 Mg/ml of chloramphenicol, and incu- chamber was graded according to the scheme of bated at room temperature in microtiter wells. After 1 Schlaegel,5 and inflammation of the vitreous space hr, live microfilariae that had emerged from the speci- was graded according to the scheme of Kimura et al.6 mens were washed in RPMI-1640, filtered in a sterile Blood samples drawn on day 53 were lost due to an column containing a fine cellulose sponge, centri- equipment failure. On day 53 the animals were killed fuged at 400 X g, and resuspended in distilled water to under pentobarbital anesthesia, and the eyes were remove residual erythrocytes by osmotic shock. They preserved in buffered glutaraldehyde and processed were then washed with RPMI-1640, centrifuged for histopathology. Serial sections in the plane of the twice, and resuspended to Dulbecco's phosphate- pupil, optic nerve head, and macula of each eye were buffered saline (PBS). To prepare dead microfilariae prepared. for intravitreal injection, live microfilariae in PBS Enzyme-linked immunosorbent assays for immu- were submerged suddenly in liquid nitrogen, then im- noglobulin (Ig) G antibody were done in triplicate us- mediately thawed. All microfilariae were observed to ing Immulon 2 microplates (Dynatech, Alexandria, be dead after this freeze-thaw treatment. VA) coated with 1 fig of O. volvulus adult worm ex- Four colony-raised cynomolgus monkeys (Macaca tract per well. Plates were incubated with 10% normal fascicularis) were used in the protocol as shown in goat serum in PBS for 1 hr at room temperature, with Table 1. In a separate dose-determining experiment monkey sera at 1:100 dilution for 2 hr at 37°C, and with four other colony-raised cynomolgus monkeys with peroxidase-conjugated goat anti-human IgG (unpublished), we found the optimal intravitreal dose (Tago, Burlingame, CA) for 2 hr at 37°C. The sub- to be 10,000 live O. volvulus microfilariae. Because strate was developed as previously described:2 O. vol- the intraocular reaction to intravitreal injection of vulus adult worms were obtained by collagenase di- dead microfilariae was not known, doses of 1000 and gestion of onchocercal nodules,7 and a crude adult 10,000 were used. Two of the four cynomolgus mon- worm extract was prepared by homogenization of the keys were first sensitized with subcutaneous injec- worms in 0.01 M PBS, pH 7.4, and centrifugation of tions of 30,000 live microfilariae followed by another the homogenate at 16,000 X g for 2 hr. subcutaneous injection of 30,000 microfilariae 2 Cell-mediated immune responses were studied us- weeks later. Each inoculum was distributed on four ing peripheral blood mononuclear leukocytes abdominal sites and given under ketamine anesthesia. (PBML) as previously described.8 These cells were Ten days after sensitization, both sensitized and un- isolated by Ficoll-Hypaque density-gradient centrifu- sensitized monkeys received an intravitreal injection gation (Sigma, St. Louis, MO) and resuspended at 1.5 of either live or dead microfilariae as in Table 1. Con- X 106/ml in minimum essential medium supple- trol intravitreal injections of PBS alone did not elicit mented with 25 mM HEPES buffer, 2 mM glutamine, any chorioretinal changes2-3 and therefore were not 100 U/ml penicillin, 100 fig/m\ streptomycin, and used in the protocol. A 1-ml tuberculin syringe with a 10% fetal bovine serum (all Gibco). Concanavalin A 30-gauge needle was used to inject the 0.1-ml volume (Sigma, St. Louis, MO) was used as a initogen at a containing the microfilariae. Anterior chamber para- concentration of 400 Mg/ml (100 /tg/well) in complete medium. Proliferation assays were done in round- bottom microtiter wells (Linbro, McLean, VA) with 5 Table 1. Experimental protocol 1.5 X 10 PBML and 100 ^g of antigen or mitogen in a final volume of 200 n\. Control wells contained 100 n\ Animal Sensitized Eye Intravitreal mf complete medium in place of antigen or mitogen. Triplicate cultures were incubated in 37°C in 5% CO2 239 Yes OD 10,000 live 3 OS 1000 dead for 3 days, at which time 1 ixd of H-thymidine was 266 Yes OD 10,000 live added to each well. Cultures were harvested 18 hr OS 10,000 dead later, and the radioactivity was counted in a liquid 331 No OD 10,000 live OS 1000 dead scintillation counter (Unilux III, Tm Analytic, Elk 369 No OD 10,000 live Grove Village, IL). Stimulation indices (SI) were cal- OS 10,000 dead culated as follows: SI = mean cpm test well/mean mf = Microfilariae. cpm control well.

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Table 2. Clinical findings of the retina and choroid

Animal DayO Day 1 Day 10 Day 35 Day 53

239 OD Normal Slight retinal Moderate pigment Moderate pigment Moderate pigment pigment changes loss temporally loss temporally loss temporally OS Normal Normal Normal Slight pigment Slight pigment mottling mottling 266 OD Normal Normal Slight pigment Slight pigment (Died) mottling mottling temporally temporally OS Normal Normal Normal Normal (Died) 331 OD Normal Normal Moderate pigment Large patchy Large patchy loss temporally pigment loss pigment loss OS Normal Normal Normal Normal Normal 369 OD Normal Small patchy Moderate patchy Moderate patchy Moderate patchy pigment loss pigment loss pigment loss pigment loss OS Normal Normal Normal Normal Normal

O. volvulus adult worm crude antigen was prepared animals showed patchy loss of retinal pigment in the in complete medium for a final concentration of 50 temporal periphery of the eyes that received live mi- and 25 jug/well. Pooled whole guinea pig retinas were crofilariae. The patches of retinal pigment loss gradu- homogenized in PBS, 0.15 NaCl, 0.01 M phosphate, ally grew larger in area from day 10 until death (Fig. pH 7.4, and centrifuged at 10,000 X g for 10 min. The 3A). Fluorescein angiography revealed patchy loss of pellet was discarded, and the supernatant frozen at -80°C. Crude retinal antigen was diluted in complete medium and used at 100 and 50 /xg/well. 239 OD (10.000 Bve) 239 OS (1000 dead) The study was conducted in accordance with the 26600 (10.000 live) ARVO Resolution on the Use of Animals in Re- 266 OS (10.000 dead) search.

Results Anterior Chamber Cells and Flare Clinical findings in the retina and choroid of the animals are summarized in Table 2. Intravitreal injection of 10,000 live microfilariae elicited chorioretinal changes resembling onchocerciasis in both sensitized and unsensitized animals. Intravitreal injection of 1000 or 10,000 dead microfilariae generally elicited much less pronounced chorioretinal changes. Inflam- mation of the anterior segment was generally more pronounced in eyes that received live microfilariae; 331 OD (10.000 Sve) these findings are summarized in Figures 1A and IB. 331 OS (1000 dead) Inflammation of the vitreous space generally ap- 369 OD (10,000 Ive) peared earlier in animals that had been sensitized 369 OS (10.000 dead) (Figs. 2A-B). Intravitreal injection of live O. volvulus microfilar-

iae in the right eyes of sensitized monkeys (#239 and Anterior Chamber #266) resulted in moderate anterior chamber cell and Cells and Flare flare which appeared by day 1 and persisted for ap- proximately 3-6 weeks after intravitreal injection. Live microfilariae were present in the anterior chamber of animal 239 until day 53 when the animals were killed. Live microfilariae were observed in the B anterior chamber of animal 266 until day 45 at which time this animal died from an unrelated cause. (Au- topsy revealed leukemic infiltrates in the bone Fig. 1. (A) Anterior chamber cell and flare in right and left eyes of sensitized animals is plotted against time. (B) Anterior chamber cell marrow.) Inflammation of the vitreous was present by and flare in right and left eyes of unsensitized animals is plotted day 10 in both animals and decreased by day 35. Both against time.

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239 00 (10,000 live) 239 OS (1000 dead) 266 OD (10.000 6ve) 266 OS (10,000 dead)

Vilreous Haze

331 OD (10.000 live) 331 OS (1000 dead) 369 OD (10,000 Sve) 369 OS (10,000 dead)

Vitreous Haze

Fig. 3. (A) Areas of retinal pigment loss in animal 239 at day 35. (B) Fluorescein angiogram of same animal shows patchy choroidal hyperfluorescence. No late leakage was noted from the choroid.

Fig. 2. (A) Vitreous haze in right and left eyes of sensitized animals is plotted against time. (B) Vitreous haze in right and left eyes and #369) resulted in a mild to moderate anterior of unsensitized animals is plotted against time. chamber cell and flare which was most pronounced at days 1 and 18 and gradually decreased. Live microfi- pigment from the retinal pigment epithelium with lariae were noted in the anterior chamber of both ani- choroidal hyperfluorescence (Fig. 3B). There was no mals after injection; these were present in animal 331 late leakage seen on fluorescein angiography from the until day 53. Mild inflammation of the vitreous was areas of choroidal hyperfluorescence. Intravitreal injection of dead micronlariae in the left eyes of sensitized monkeys (#239 and #266) resulted in mild to moderate anterior segment cell and flare; this reached a peak by days 10-18 and then dis- appeared by day 41 after injection. No microfilariae were observed in the anterior chamber. Inflammation of the vitreous was present by day 10 in the left eye of animal 266 (received 10,000 dead micronlariae). At day 18, cells were observed adjacent to the dead microfilariae in the vitreous. No inflammation of the vitreous was observed in the left eye of animal 239. Slight mottling of the pigment was present in the temporal periphery of the left eye of animal 239 by day 35, and no chorioretinal changes were observed in the left eye of animal 266. Fig. 4. Fundus of animal 331 at day 45 after intravitreal injection Intravitreal injection of live O. volvulus micronlar- of 10,000 live microfilariae. Patchy loss of retinal pigment is pres- iae in the right eyes of unsensitized monkeys (#331 ent.

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The histopathologic findings are summarized in Ta- ble 3. Foci of chronic inflammatory cells were present in the ciliary body of all eyes except the left eye of animal 331. Foci of chronic inflammation were larger and more numerous in the ciliary body of the eyes that received live microfilariae (Fig. 6) than in eyes that received dead microfilariae. Both eyes of animal 239 had thinning and loss of outer retinal layers and retinal pigment epithelium in the temporal periphery with inflammation in the overlying vitreous. In the right eye of animal 266, microfilariae were present in the vitreous with a light scattering of eosinophils, mononuclear cells, and occasional giant cells (Figs. 7A-B); in the left eye degenerating microfilariae were Fig. 5. Patchy loss of pigment with pigment clumping in tem- present with occasional multinucleated giant cells and poral periphery of unsensitized animal (#331) at day 45 following eosinophils. The areas of slight retinal pigmentary intravitreal injection of 10,000 live microfilariae. changes noted on clinical examination of the right eye of animal 266 were not confirmed by histopathology. present in animal 331 by day 1 and decreased by day The right eye of animal 331 had extensive loss of 10. Both unsensitized animals had patchy areas of outer retinal layers and retinal pigment epithelium in pigment loss occurring randomly in the nasal and the temporal periphery (Fig. 8) with aggregates of temporal retina (Fig. 4). These areas grew steadily mononuclear cells in the subjacent choroid. In the left larger from days 1-53 and were more extensive in eye there were rare chronic inflammatory cells in the area in the unsensitized animals than in the sensitized vitreous and the retina and choroid were normal. In animals. Retinal pigment clumping in the temporal the right eye of animal 369 there was a focus of periphery was seen in both unsensitized animals by chronic inflammatory cells and eosinophils in the cho- day 35 (Fig. 5), and live microfilariae were observed roid and subjacent retinal pigment epithelium; in the transiently in the retina in animal 369. When live mi- left eye there was a light scattering of chronic inflam- crofilariae were observed in the retina, the surround- matory cells and eosinophils in the vitreous, and the ing retina appeared normal. retina and choroid were normal. No microfilariae Intravitreal injection of dead microfilariae into the were found in the retina, choroid, or sclera of any of left eyes of unsensitized animals (#331 and #369) re- the animals. In general, pathologic changes in the ret- sulted in slight to moderate anterior chamber cell and ina and choroid were relatively more pronounced in flare; this was most pronounced at days 10-18 and eyes that received live microfilariae than in those that decreased thereafter. No microfilariae were observed received dead microfilariae. in the anterior chamber. A moderate vitritis was pres- Sensitization of systemically immunized monkeys ent in animal 369 that was maximum at days 10 and was confirmed by detection of IgG to onchocercal mi- 18. No inflammation of the vitreous was observed in crofilarial antigens in serum by enzyme-linked im- the left eye of animal 331. No clinical changes were munosorbent assay (Fig. 9). Both sensitized animals observed in the retina and choroid of the left eye of (#239 and #266) had an IgG titer 10 days after receiv- both animals. Dead microfilariae were observed in ing the second subcutaneous injection of microfilar- the vitreous and adjacent to normal-appearing retina. iae. Both unsensitized monkeys had an IgG titer

Table 3. Summary of histopathologic findings

Inflammation, Inflammation, Thinning, loss. Retinal Pigment Inflammation, Animal Eye ciliary body vitreous outer retina hemorrhage migration choroid

239 OD ++ +++ + ++ +++ OS + + +++ ++ 266 OD ++ ++ (mf) OS + ++(mf) 331 OD +++ ++ .+. OS + + (mf) 369 OD +++ + OS ++ ++

mf = Microfilariae present.

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Fig. 6. Large focus of chronic inflammatory cells in the ciliary body with light scattering of chronic inflammatory cells in the vitreous at day 46 in sensitized animal (#266) which received intravitreal injection of 10,000 live microfilariae.

Fig. 7. (A) Microfilaria in the posterior vitreous with light scattering of chronic inflammatory cells and eosinophils in right eye of animal (#266) which received live microfilariae. (B) High-power magnifica- tion of cells in the vitreous showing mononuclear cells, eosinophils, and a multinucleated giant cell.

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Fig. 8. Extensive loss of outer retinal layers with migration of pigmented cells into the inner retina in unsensitized animal (#331) which received intravitreal injection of 10,000 live microfilariae.

within the first 3 weeks after intravitreal injection of chorioretinal changes which resemble human oncho- microfilariae, indicating they were sensitized by this cercal chorioretinitis, and these changes more closely route. resemble the human disease than the primate model Cell-mediated immune responses to O. volvulus that uses O. lienalis microfilariae. In this primate adult worm crude antigen are shown in Figure 10. model, live microfilariae were observed in the ante- Sensitized animals produced positive T-cell responses rior chamber for many weeks, and they were observed at the time of intravitreal injection, and the magni- in two of the animals until they were killed. Anterior tude of these responses continued to increase over the chamber inflammation was present in primate eyes next 18 days. Unsensitized animals had no apparent that received intravitreal injections of live or dead mi- T-cell response at the time of intravitreal injection but crofilariae. Live microfilariae are often present in the became positive 18 days after injection, again demon- anterior chamber in human ocular onchocerciasis, strating that they had become sensitized by this route. but usually there is no accompanying cell and flare. None of the animals had a significant response to Some humans have significant iridocyclitis in ocular crude retinal antigen at any time, and lymphoprolifer- onchocerciasis, and this appears to be similar to the ative responses to mitogen were normal in all the ani- anterior segment inflammation in the primate model. mals throughout the experiment (data not shown). The introduction of a large number of microfilariae into the primate eye at one time may cause the in- Discussion We demonstrated that live O. volvulus microfilariae injected into the vitreous of primates can produce

239 (ImnunEzed) 11D0- 266 (Invrunlzod) 1000- Wl(NoHmmunct

STIMULATION INDEX

EL1SA M92K1000

soo- 100- 14 81 it 35

Oays

t Days nwti Fig. 10. Cell-mediated immune responses against O. volvulus Fig. 9. IgG activity against O. volvulus microfilarial antigen. adult antigen. Stimulation index (CPM [3H]-thymidine incorpora- ELISA A492 X 1000 for serum at 1:500 dilution is plotted against tion with antigen/incorporation without antigen) is plotted against time. Horizontal dashed line denotes minimum positive (mean for time. Dashed line indicates minimum positive value (two-fold stim- normal serum plus 3 X SD). ulation above background).

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creased amount of inflammation in the anterior in the primate model, it may be necessary to repeat chamber. intravitreal injections of live microfilariae. Intravitreal injections of O. volvulus microfilariae We examined the IgG response to onchocercal anti- resulted in progressive loss of retinal pigment and pig- gen and systemic cell-mediated proliferation re- ment clumping, and these two clinical features are sponses to mitogen and onchocercal microfilarial an- also characteristic of human ocular onchocerciasis. tigen to correlate these responses with the develop- The major difference between the posterior segment ment of posterior segment lesions. Systemic IgG changes in the primate model and human disease was responses in unsensitized animals occurred within 3 the more rapid progression of lesions in the animal weeks of the intravitreal injection. Cell-mediated im- model. The primate model may be an accelerated ver- mune responses to onchocercal microfilarial antigen sion of human onchocerciasis because the numbers of were present in all animals by 18 days after intravit- microfilariae are probably much greater than present real injection. The cell-mediated responses to Oncho- in human disease. Posterior segment disease in the cerca were the smallest in animal 266, suggesting that primate eye evolved over several weeks, whereas hu- the animal, who died of leukemia, had reduced con- man onchocercal posterior segment disease appears centrations of lymphocyte responders. Areas of reti- to evolve over several years.9 nal pigment loss developed within the first day after O. volvulus microfilariae produce a more accurate intravitreal injection of live microfilariae in this ex- model of human ocular onchocerciasis than O. liena- periment and gradually grew more severe. Immuno- lis in primates because there is less ocular inflamma- logic sensitization, as demonstrated by circulating tion in both the anterior and posterior segments. In IgG antibody and cell-mediated immunity, did not our study, intravitreal injections of 10,000 live O. vol- appear to correlate with the early development of cho- vulus microfilariae produced less inflammation than rioretinal lesions. Since pathologic specimens of the intravitreal injections of 500 live O. lienalis microfi- eye were not available from the first few weeks of the lariae. In previous studies with O. lienalis, intravitreal animal model, the extent of immune-mediated mech- injection was followed by fibrin clot formation and anisms in the development of chorioretinal lesions even severe iritis with hypopyon,3-4 whereas only a cannot be stated definitely. An apparent lack of corre- moderate anterior chamber cell and flare were noted lation between circulating IgG or IgE antibody, local in our study with O. volvulus. Chorioretinal changes IgG antibody, cell-mediated immunity, and clinical in our study were characterized by retinal pigment disease has also been previously observed in our ear- loss rather than pronounced retinal vasculitis and pig- lier studies with O. lienalis, suggesting that local in- ment loss seen with O. lienalis. O. volvulus appears to flammatory mechanisms may be of primary impor- be less antigenic in the primate model than O. liena- tance in the early development of lesions in the poste- 4 lis, the species causing bovine onchocerciasis. rior segment of the eye. Sensitization may have Both live and dead O. volvulus microfilariae were inhibited later development of patchy areas of retinal injected intravitreally to determine whether direct in- pigment loss; the lesions were more extensive in ani- vasion of the retina and choroid by microfilariae mals that had not been previously sensitized. Our would be necessary to produce chorioretinal lesions. previous studies in the monkey model also showed that unsensitized animals had more extensive disease Overall, chorioretinal changes were more pro- 3 nounced in eyes that received live microfilariae, sug- than sensitized animals. The cell-mediated prolifera- gesting the passage of microfilariae through the retina tive response to crude retinal antigen was examined and choroid may play a role in the pathogenesis of because autoimmunity against the retina has been onchocercal posterior pole disease. However, one ani- suggested to play a role in the retinal lesions." Cell- mal (#239) had thinning and loss of the outer retina in mediated immune responses against the retina were both eyes, suggesting that the presence of dead micro- not present in any animal in this study. filariae alone can contribute to chorioretinal changes. Further studies in this model may aid in under- Histopathologic examination of all the eyes did not standing the disease processes involved and the devel- reveal microfilariae in the retina and choroid, al- opment of the devastating chorioretinal lesions which though in previous experiments, within 1 week of in- are a major cause of blindness in onchocerciasis. travitreal injection, live microfilariae were found in 3 Key words: onchocerciasis, Onchocerca, chorioretinitis, an- the retina. In a murine model, onchocercal microfi- imal model, microfilaria lariae migrated rapidly through ocular tissues within hours to days.10 In our study, it appears that after 53 Acknowledgments days, most of the live microfilariae may have mi- The authors thank Drs. Noel Williams and John Boateng grated out of the eye or died. This suggests that, to of the Liberian Agricultural Company, Keene Industries, continue the progression of posterior segment disease Grand Bassa County, Liberia, Dr. Henry Newland, now at

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International Community Eye Care Center, Flinders Medi- munity in the chorioretinitis elicited by intravitreal Oncho- cal Centre, Bedford Park, Australia, and Betsy Brotman, cerca lienalis microfilariae. Trop Med Parasitol 39:111, 1988. Liberian Institute for Biomedical Research, Robertsfield, 5. Schlaegel TF Jr: Symptoms and signs of uveitis. In Clinical Liberia, for medical assistance, and Mr. Sam D'Anna, Ophthalmology, Vol 4, Duane TD, editor. Philadelphia, Wilmer Institute, Johns Hopkins University, for expert Harper and Row, 1983, pp. 1-7. technical assistance. 6. Kimura SJ, Thygeson P, and Hogan MJ: Signs and symptoms of uveitis: II. Classification of the posterior manifestations of uveitis. Am J Ophthalmol 47:171, 1959. References 7. Schulz-Key H, Albiez EJ, and Buttner DW: Isolation of living adult Onchocerca volvulus from nodules. Trop Med Parasitol 1. World Health Organization Expert Committee on Onchocer- 28:428, 1977. ciasis. Third Report, WHO Technical Report Series 752. Ge- 8. Young E, Schachter J, Prendergast RA, and Taylor HR: The neva, World Health Organization, 1987, p. 167. effect of cyclosporine in chlamydial eye infection. Curr Eye Res 2. Donnelly JJ, Taylor HR, Young EM, Khatami M, Lok JB, and 6:683, 1987. Rockey JH: Experimental ocular onchocerciasis in cynomol- 9. Semba RD, Murphy RP, Newland HS, Awadzi K, Greene BM, gus monkeys. Invest Ophthalmol Vis Sci 27:492, 1986. and Taylor HR: Longitudinal study of the posterior segment 3. Semba RD, Donnelly JJ, Rockey JH, Lok JB, Sakla AA, and lesion of onchocerciasis. Ophthalmology 97:1334-1341, 1990. Taylor HR: Experimental ocular onchocerciasis in cynomol- 10. James ER, Smith B, and Donnelly J: Invasion of the mouse eye gus monkeys: II. Chorioretinitis elicited by intravitreal Oncho- by Onchocerca microfilariae. Trop Med Parasitol 37:359, cerca lienalis microfilariae. Invest Ophthalmol Vis Sci 1986. 29:1642, 1988. 11. Chan CC, Nussenblatt.RB, Kim MK, Palestine AG, Awadzi K, 4. Donnelly JJ, Semba RD, Xi MS, Young E, and Rockey JH: and Ottesen EA: Immunopathology of ocular onchocerciasis: Experimental ocular onchocerciasis in cynomolgus monkeys: 2. Anti-retinal autoantibodies in serum and ocular fluids. Oph- III. Roles of IgG and IgE autoantibody and cell-mediated im- thalmology 94:439, 1987.

CORRECTION In the article, "Relations Between Fundus Appearance and Function: Eyes Whose Fellow Eye Has Exudative Age-Related Macular Degeneration," by Alvin Eisner, Vasiliki D. Stoumbos, Michael L. Klein, and Susan A. Fleming, which appeared in the January 1991 issue of Investigative Ophthalmology and Visual Science (pages 8-20), the third category in the Appendix is labeled "Reticular Drusen Area." The correct label should read "Drusen Area." "Reticular" should be the last entry under "Drusen Size." The publisher regrets any inconvenience this error may have caused.

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