Experimental Ocular Onchocerciasis in Cynomolgus Monkeys

Transcription

1 Investigative Ophthalmology & Visual Science, Vol. 29, No. 11, November 1988 Copyright Association for Research in Vision and Ophthalmology Experimental Ocular Onchocerciasis in Cynomolgus Monkeys //. Chorioreriniris Elicited by Inrrovirreol Onchocerca lienalis Microfi/ariae Richard D. Semba,* John J. Donnelly,t John H. Rockey.f James B. Lok4 Aref A. Saklat and Hugh R. Taylor* Chorioretinitis due to onchocerciasis is a major cause of blindness, and the pathogenesis is poorly understood. We have developed an experimental model for onchocercal chorioretinitis using cynomolgus monkeys (Macaca fascicularis). Two normal monkeys and two monkeys which had received prior sensitization with subcutaneous injections of live Onchocerca lienalis microfilariae were given intravitreal injections of either 0,10, 50 or 500 live microfilariae. Posterior segment changes included disc edema, venous engorgement, retinal vasculitis, intraretinal hemorrhage, and progressive retinal pigment epithelial (RPE) disturbances. Histopathological findings included perivascular infiltrates with eosinophils, eosinophilic choroiditis, and RPE hypertrophy, hyperplasia and loss of pigment. Microfilariae in the retina had no surrounding inflammation but were found adjacent to areas of RPE alterations. Overall the inflammatory reaction in the two unsensitized monkeys was more severe than that seen in the sensitized monkeys. The retinal appearance of the monkeys resembled that found in human onchocerciasis, and this model appears to be a promising one for future investigations. Invest Ophthalmol Vis Sci 29: ,1988 Onchocerciasis, a filarial infection caused by Onchocerca volvulus, is a major cause of blindness in endemic areas of Africa and Central and South America. Much of the blindness from onchocerciasis is due to a characteristic, progressive chorioretinal scarring, 1 but the pathogenesis of this fundus lesion is unclear. 2 It is difficult to obtain affected human eyes for histopathological study, and the few eyes which have been studied exhibit far advanced disease with other accompanying pathology. Thus, the pathogenesis of the extensive chorioretinal damage in onchocerciasis cannot be easily determined from human material. From The International Center for Epidemiologic and Preventive Ophthalmology, The Wilmer Institute and the School of Hygiene and Public Health, The Johns Hopkins University, Baltimore, Maryland, the fdepartment of Ophthalmology, Scheie Eye Institute, University of Pennsylvania School of Medicine, and the ^Department of Pathobiology, University of Pennsylvania School of Veterinary Medicine, Philadelphia, Pennsylvania. Supported in part by NIH Grant EY (HRT), EY (RDS), EY (JHR), EY (JJD); and AI19995 (JBL) and by grants from the World Health Organization Special Program for Research and Training in Tropical Diseases (RDS) and Onchocerciasis Control Programme Onchocerciasis Chemotherapy Project (JJD). Submitted for publication: March 5, 1987; accepted June 13, Reprint requests: Dr. Hugh R. Taylor, The Wilmer Institute, The Johns Hopkins Hospital, Baltimore, MD Experimental animal models have provided additional information on the immunopathology and pathogenesis of ocular onchocerciasis. 3 Chorioretinal lesions have been produced by intraorbital implantation of O. volvulus adult worms 4 and intravitreal and subretinal injections of O. volvulus microfilariae in the rabbit. 5 ' 6 Because O. volvulus microfilariae can only be obtained from humans and are difficult to obtain in large quantity, the alternative use of Onchocerca spp. microfilariae from other animals, such as O. cervicalis from the horse and O. gutturosa and O. lienalis from cattle, has been explored. 3 The use of closely related species of microfilariae appears to be a good alternative in animal models. The nonhuman primate eye is a more accurate anatomical model than the lagomorph or rodent eye for producing lesions resembling human onchocerciasis. In a recent study of experimental ocular onchocerciasis in cynomolgus monkeys, intravitreal injections of approximately 10,000 O. lienalis microfilariae led to an intense posterior and anterior chamber reaction which precluded a view of the fundus. 7 The purpose of the present study is to determine whether the posterior segment lesions of onchocerciasis can be reproduced in cynomolgus monkeys by intravitreal injection of small numbers of microfilariae. This would more closely resemble the clinical situation, since both histopathological studies 8 and clinical observations 9 have shown that microfilariae 1642

2 No. 11 EXPERIMENTAL ONCHOCERCAL CHORIORETINITIS / Sembo er ol 1643 Table 1. Clinical findings in cynomolgus monkeys receiving intravitreal O. lienalis microfilariae Monkey Sensitized* Intravitreal Microfilariae] Anterior chamber Clinical status 1 week after inoculation Vitreous Retina/choroid yes yes no no 500 O.D. 50 O.S. 10 O.D. OO.S. (balanced salt solution) 500 O.D. 50 O.S. 10 O.D. OO.S. (balanced salt solution) Small hypopon Small fibrin clot Mild anterior uveitis Hyphema, fibrin clot Small fibrin clot Mild anterior uveitis Moderate Moderate Trace Moderate Mild Mild Large patchy RPE pigment loss, leakage on fluorescein Trace vascular sheathing and mild disc edema Small intraretinal hemorrhages and trace disc edema Large patchy RPE pigment loss, small intraretinal hemorrhages, mild disc edema Large intraretinal hemorrhages, vasculitis, patchy RPE pigment loss, mild disc edema Moderate hemorrhagic vasculitis with sheathing * 23,000 O. lienalis microfilariae injected subcutaneously 14 days plus 30,000 microfilariae injected subcutaneously 7 days prior to intravitreal challenge. t Intravitreal inoculation of microfilariae on day 0. are only occasionally encountered in the vitreous, retina, and choroid, in spite of the extensive chorioretinal damage which has been observed. In addition, in vitro studies have suggested that onchocercal antigens may suppress the cellular immune response to infection by Onchocerca, and thus the role of sensitization on the subsequent ocular response was investigated. This study suggests that chorioretinal lesions resembling those of ocular onchocerciasis may be produced by small numbers of microfilariae. Materials and Methods Microfilariae of O. lienalis were obtained from the umbilical skin of cattle by the method of Bianco 12 and were cryopreserved using ethanediol. 13 Motility of cryopreserved microfilariae was noted to be 90% or greater. Cryopreserved microfilariae were prepared for injection by thawing in Tyrode's solution with 20% fetal bovine serum (FBS), penicillin (100 units/ ml), and streptomycin (100 fig/ml) warmed to 38 C; washing three times in cold Tyrode's solution with antibiotics but without FBS; and then being used immediately for injection. Two of four cynomolgus monkeys (Macaca fascicularis) were sensitized with one subcutaneous injection of 23,000 O. lienalis microfilariae followed by another subcutaneous injection of 30,000 microfilariae 1 week later. Each inoculum was distributed on four sites on the abdomen and given under ketamine anesthesia. Peripheral blood mononuclear leukocyte (PBML) proliferative responses to O. volvulus adult worm crude extract antigen have been demonstrated to increase five- to eight-fold following sensitization with similar doses of O. lienalis in other experiments (unpublished). After sensitization, IgG antibody titers to microfilarial extract antigen were determined by enzyme-linked immunoabsorbent assay (ELISA) as previously described. 7 One week following the second inoculation, the sensitized and unsensitized monkeys received an intravitreal injection of either 0 (control injection), 10, 50, or 500 microfilariae in balanced salt solution through a pars plana approach (Table 1). A 1 ml tuberculin syringe with a 30 gauge needle was used to inject the 0.1 ml volume which contained microfilariae. Anterior chamber paracentesis was performed after the intravitreal injections to relieve intraocular pressure.

3 1644 INVESTIGATIVE OPHTHALMOLOGY b VISUAL SCIENCE / November 1988 Vol. 29 Fig. 1. (A) Vasculitis and intraretinal hemorrhages in an eye of a naive monkey which received ten micronlariae in the vitreous. (B) Fluorescein angiogram of the same fundus demonstrating leakage from terminal venules. Clinical slit-lamp examination, anterior segment photography, fundus photography, and fluorescein angiography were performed 1 day prior to intravitreal injection and on the third and seventh days following injection. Ocular inflammation was graded according to the scheme of Schlaegel. 14 All monkeys were sacrificed on day 7 after intravitreal injection, and the eyes were preserved in glutaraldehyde and processed for light and electron microscopy. The study was conducted in accordance with the ARVO Resolution on the Use of Animals in Research. Results The inflammatory reaction in the two unsensitized monkeys was overall more severe than that seen in the two sensitized monkeys. Both the degree and duration of anterior uveitis and chorioretinitis was greater in unsensitized monkeys (Table 1). Sensitization of systemically immunized monkeys was confirmed by detection of IgG antibody to microfilarial antigens in serum by ELISA. Preimmunization ELISA absorptivities were and 0.032, while postimmunization absorptivities were and in animals 1 and 2, respectively. Unsensitized monkeys had absorptivities of and (animals 3 and 4) at the time of intravitreal injection of micronlariae. Intravitreal injection of micronlariae in unsensitized monkeys resulted in a marked anterior uveitis which varied from a fibrin clot with keratoprecipitates in the eye which received 50 micronlariae to a more severe reaction which included a small hyphema in the eye which received 500 micronlariae. Posterior segment changes in the eye of the unsensitized monkey which received ten intravitreal micronlariae included mild disc edema with venous engorgement and a severe retinal vasculitis with sheathing, adjacent intraretinal hemorrhages, but only minimal (Fig. 1 A). Fluorescein angiogram revealed diffuse leakage along inflamed retinal vessels, especially terminal venules (Fig. IB). The control eye remained normal. The unsensitized monkey which received 50 intravitreal micronlariae developed an extensive hemorrhagic vasculitis with sheathing in the area temporal to the macula (Fig. 2A). A mild was present with mild disc edema and venous engorgement. Loss of pigment was present in small focal areas and appeared as choroidal hyperfluorescence with leakage onfluoresceinangiogram (Fig. 2B, C). By day 7, the small areas which exhibited pigment loss became enlarged and confluent (Fig. 2D) and continued to show choroidal hyperfluorescence and leakage (Fig. 2E). Intravitreal injection of 500 micronlariae in an unsensitized monkey eye resulted in a moderate, mild disc edema with venous engorgement, and small intraretinal hemorrhages temporal to the macula. By day 7, large focal areas of retinal pigment loss were noted temporal to the macula and showed marked leakage. Intravitreal injection of micronlariae in the sensitized monkeys resulted in an anterior uveitis which ranged from a mild reaction in the eye which received ten micronlariae to a small hypopyon in the eye which received 500 micronlariae. In general, the anterior uveitis was most pronounced on the first day after intravitreal injection and gradually resolved toward day 7. In the sensitized monkey, injection often micronlariae resulted in a mild, mild disc edema with venous engorgement, and two small intraretinal hemorrhages in the superotemporal midperiphery. On day 3, fluorescein angiogram demonstrated mild disc leakage which resolved by day 7. The control eye remained normal. Injection of 50 micronlariae in the sensitized monkey resulted in a mild, mild disc edema and venous engorgement, and trace vascular sheathing on day 3. By day

4 No. 11 EXPERIMENTAL ONCHOCERCAL CHORIORETINITIS / Semba er al 1645 Fig. 2. (A) Vasculitis, intraretinal hemorrhage, and RPE changes at day 3 in an eye of a naive monkey which received 50 microftfariae. (B, C) Fluorescein angiogram at day 3 demonstrating leakage from choroid in area of RPE pigment loss (arrow). (D) By day 7 the vasculitis was resolving and the areas of pigment loss from the RPE grew large and confluent. (E) Fluorescein angiogram on day 7 which shows choroidal hyperfiuorescence. 7, no vascular sheathing was seen, and an area of focal was noted temporal to the macula. In the other eye, the injection of 500 microfilariae caused a moderate with two patchy areas of pigment loss temporal to the macula. By day 7, the areas of pigment loss became large and confluent (Fig. 3); and on fluorescein angiogram, the area appeared as retinal pigment epithelial (RPE) transmission defects with leakage (Fig. 4). Fluorescein angiogram on day 3 showed small punctate areas of choroidal hyperfiuorescence with leakage. These focal areas later appeared as small RPE defects by day 7. Histologic studies of eyes from unsensitized monkeys which were injected with ten microfilariae showed mild vasculitis with eosinophils and intraretinal hemorrhages, and the eyes which received 50 and 500 microfilariae showed extensive intraretinal hemorrhages, perivascular inflammatory cell infiltrates, and loss of pigment and vacuolation of the RPE (Fig. 5). Retinal arteries in the midperipheral retina had perivascular infiltrates of eosinophils (Fig. 6). Retinal edema and perivascular infiltrates were present along retinal venules. Mild vitreous hemorrhages with fibrin and enmeshed eosinophils, neutrophils, and

5 1646 INVESTIGATIVE OPHTHALMOLOGY 6 VISUAL SCIENCE / November 1968 Vol. 29 Fig. 3. Large area of pigment loss from the RPE in an eye of an immunized monkey which received 500 microfilariae in the vitre- Fig. 4. Fluorescein angiogram of the same fundus shown in Figure 3., demonstrating RPE transmission defects with choroidal hyperfluorescence. macrophages were present in these eyes. Focal choroidal inflammatory cell infiltrates consisting of eosinophils and plasma cells were present in a patchy distribution throughout the posterior segment. Retina adjacent to the choroidal microgranulomas showed RPE hypertrophy and hyperplasia and pathologic alterations in the other retina (Fig. 7). Pathologic findings in the sensitized monkeys included ini > Fig. 5. Pigment loss and duplication of the RPE in a naive monkey which received 50 intravitreal microfilariae (magnification X571).

6 No. 11 EXPERIMENTAL ONCHOCERCAL CHORIORETINITIS / Sembo er ol 1647 Fig. 6. Retinal artery in midperipheral retina showing perivascular infiltrate of eosinophils (magnification X578). traretinal hemorrhages in the eye which received ten micronlariae, perivasculitis with eosinophils in the eye which received 50 micronlariae (Fig. 8), and extensive eosinophilic perivasculitis and choroiditis in the eye which received 500 micronlariae. Micronlariae were found in the retina which had no surrounding inflammatory infiltrates but were located adjacent to areas of RPE hypertrophy and hyperplasia (Fig. 9). Discussion The presence of live Onchocerca micronlariae in the vitreous of the cynomologus monkey eye produces chorioretinal lesions which resemble those seen in human ocular onchocerciasis. This suggests that posterior pole lesions may be directly related to the presence of micronlariae. Many theories for the pathogenesis of the posterior pole lesions of onchocerciasis have been advanced since they were first described by Hissette Bryant 17 suggested that the fundus lesions were caused by a circulating toxin secreted by adult worms. In a review of the previous studies by Hissette and Bryant and an additional case report, 18 Ridley 19 concluded that dead microfilariae provoke an inflammatory reaction with occlusion of smaller blood vessels. He suggested that perivascular infiltration and endothelial proliferation contributed to the chorioretinal degeneration. It also has been speculated that deficiency of a B complex vitamin played an etiological role. 20 The circulating toxin theory was further supported by Budden, 21 who noted that the lesions did not correlate with the intensity of infection, and by others based on histopathological study Choyce 24 believed that the fundus lesion was not related to onchocerciasis at all but was rather a hereditary tapetoretinal degeneration, a widely disputed view ' 26 Several cases of florid posterior exudative uveitis have been observed in onchocerciasis. 27 The histopathology from enucleated eyes revealed retinal hemorrhages and RPE degeneration, hypertrophy and hyperplasia near microfilariae. 27 Rodger believed that

7 1648 INVESTIGATIVE OPHTHALMOLOGY b VISUAL SCIENCE / November 1988 Vol. 29 -<v Fig. 7. Choroidal microgranulomas with adjacent RPE pathologic alterations and loss of normal retinal architecture (magnification X247). active posterior uveitis was a distinct entity due to the presence of microfilariae and that the usual chorioretinal lesions associated with onchocerciasis were due to vitamin A deficiency combined with a circulating toxin from adult worms. 28 Subsequent studies have not shown any evidence that systemic vitamin A deficiency plays any role in the disease.' 293 A histopathological study of the typical advanced posterior pole lesion showed microfilarial debris within areas of posterior pole inflammation and live microfilariae in the atrophied retina. 31 There was no histologic evidence of vascular occlusion to support the mechanism postulated earlier by Ridley. 19 More recently, Greene and coworkers 32 have postulated that circulating immune complexes may play a role in the chorioretinal changes of onchocerciasis. Antiretinal antibodies are present in patients with onchocercal chorioretinitis, 3334 but the significance of circulating antibodies to retinal tissue is unclear. Recent clinical observations of patients with onchocerciasis revealed live intraretinal microfilariae and intraretinal hemorrhages, cotton wool spots and vasculitis It is not known whether this low level inflammation is related to subsequent chorioretinal changes, but both clinical and histopathological observation suggest that active infiltration of the retina by microfilariae may chronically and progressively lead to widespread chorioretinal damage. The intraretinal hemorrhages, vasculitis and RPE loss seen in the present study resemble the onchocercal lesions seen in humans. The degree of inflammation in these monkey eyes was generally greater than that seen in human onchocerciasis. A small hypopyon or fibrin clot in the anterior segment is not typical for human ocular onchocerciasis. The degree of retinal inflammation observed clinically is greater in the animal model than in the human disease. It is possible that O. lienalis, the species causing bovine onchocerciasis, is more antigenic to primates than O. volvulus, thus accounting for the greater severity of disease. A difficulty inherent in creating an animal model for human onchocercal chorioretinitis is that the human disease appears to be characterized by chronic, insidious chorioretinal damage with low-level inflamma-

8 No. 11 EXPERIMENTAL ONCHOCERCAL CHORIORETINITIS / Semba er ol 1649 fr Fig. 8. Eosinophilic perivascular infiltrate around the central retinal artery and vein in the optic disc (magnification X368). tion and the damage occurs over many years. It would be difficult to study an animal model which had the same slow progression. Our observations suggest that even small numbers of live microfilariae in the vitreous can lead to chorioretinal damage. As few as ten microfilariae in the vitreous were sufficient to cause chorioretinitis with retinal vasculitis, choroiditis and pathologic alterations in the RPE and outer retina. In this animal model, chorioretinal damage appeared to occur both from inflammation associated with the retinal vasculature and inflammation associated with the choriocapillaris. The main feature of human eyes affected by ocular onchocerciasis appears to be choroiditis and atrophy of the choroid, 831 and retinal vasculitis does not appear to be important in the pathogenesis of the posterior pole lesion. The retinal vasculitis seen in the animal model may have been much more pronounced than in the human disease because live microfilariae were injected directly into the vitreous, and the vitreous may not be the usual route for microfilariae in the eye. Chorioretinal damage may occur from host reaction to excretory-secretory antigens deposited by live microfilariae or a reaction to dead or dying microfilariae. Eosinophil granule major basic protein or eosinophil-derived neurotoxin, which may be toxic to the retina and choroid, could be released by accumulated eosinophils and elicit pathologic alterations. 36 Inflammation of the choroid with subsequent choroidal atrophy could lead to outer ischemic atrophy of the retina. The differences in the ocular response to microfilariae between the sensitized and unsensitized monkeys were pronounced in that the unsensitized monkeys developed a more severe reaction with a hemorrhagic retinal vasculitis. This suggests that previous exposure to systemic microfilariae may possibly downgrade the subsequent ocular response to microfilariae. Further studies are needed to determine the longterm effect of intravitreal microfilariae on the retina, choroid and optic nerve and to identify the immuno-

9 1650 INVESTIGATIVE OPHTHALMOLOGY & VISUAL SCIENCE / November 1988 Vol. 29 Fig. 9. Microfilariae of O. lienalis in the retina. Adjacent RPE shows pathologic alterations including duplication, pigment loss, and migration of pigment containing cells into the inner retina (magnification X571). pathological mechanisms involved in the pathogenesis of the posterior segment changes. Key words: onchocerciasis, Onchocerca, chorioretinitis, animal model, microfilaria Acknowledgments The authors wish to acknowledge and thank Shirley Johnson and Sam D'Anna for technical assistance in the project and Inga Jackman and Alice Flumbaum for help inpreparation of the manuscript. References 1. Bird AC, Anderson J, and Fuglsang H: Morphology of posterior segment lesions of the eye in patients with onchocerciasis. Br J Ophthalmol 60:2, Taylor HR: Report of a workshop: research priorities for immunologic aspects of onchocerciasis. J Infect Dis 152:389, Donnelly JJ, Rockey JH, Taylor HR, and Soulsby EJL: Onchocerciasis: experimental models of ocular disease. Rev Infect Dis 7:820, Lagraulet MJ: Chorio-retinite onchocerquienne experimentale chez le lapin. Bull Soc Ophthalmol Fr 58:8, Duke BOL and Garner A: Fundus lesions in the rabbit eye following inoculation of Onchocerca volvulus microfilariae into the posterior segment: 1. The clinical picture. Trop Med Parasitol 27:3, Garner A and Duke BOL: Fundus lesions in the rabbit eye following inoculation of Onchocerca volvulus microfilariae into the posterior segment: II, Pathology. Trop Med Parasitol 27:19, Donnelly JJ, Taylor HR, Young EM, Khatami M, LokJB, and Rockey JH: Experimental ocular onchocerciasis in cynomolgus monkeys. Invest Ophthalmol Vis Sci 27:492, Paul EV and Zimmerman LE: Some observations on the ocular pathology of onchocerciasis. Hum Pathol 1:581, Murphy RP, Taylor HR, and Greene BM: Chorioretinal damage in onchocerciasis. Am J Ophthalmol 98:519, Greene BM, Gbakima AA, Albiez EJ, and Taylor HR: Humoral and cellular immune responses to Onchocerca volvulus infections in humans. Rev Infect Dis 7:789, Greene BM, Fanning MM, and Ellner JJ: Non-specific suppression of antigen-induced lymphocyte blastogenesis in Onchocerca volvulus infection in man. Clin Exp Immunol 52:259, Bianco AE, Ham P, El Sinnary K, and Nelson GS: Large-scale recovery of Onchocerca microfilariae from naturally infected cattle and horses. Trans R Soc Trop Med Hyg 74:109, Ham P, Towson S, James ER, and Bianco AE: An improved technique for cryopreservation of Onchocerca microfilariae. Parasitology 83:139, 1981.

10 No. 11 EXPERIMENTAL ONCHOCERCAL CHORIORETINITIS / Sembo er ol Schlaegel TF Jr: Symptoms and signs of uveitis. In Clinical Ophthalmology, Vol. 4, Duane TO, editor. Philadelphia, Harper and Row, 1983, pp Hissette J: Memoire sur YOnchocerca volvulus "Leuckart" et ses manifestations oculaires au Congo beige. Ann Soc Belg MedTrop 12:433, Hissette J: Onchocerciasis in Africa and Central America. Am J Trop Med 18(Suppl):58, Bryant J: Endemic retino-choroiditis in the Anglo-Egyptian Sudan and its possible relationship to Onchocerca volvulus. Trans R Soc Trop Med Hyg 28:523, Semadini B: Histologischer Befund bei einem Fall von Zahlreichem Mikrofilarien beider Augen. Schweiz Med Wochenschr 73:75, Ridley H: Ocular onchocerciasis including an investigation in the Gold Coast. Br J Ophthalmol 10(Suppl):l, Hughes MH and Daley PF: Onchocerciasis in the southern Gold Coast. Trans R Soc Trop Med Hyg 45:243, Budden FH: Incidence of human infection with onchocerciasis in different communities in relation to the incidence and type of the ocular lesions. Br J Ophthalmol 39:321, Lavier G, Lagraulet J, and D'Haussy R: Etude anatomo-pathologique d'un oeil presentant une chorio-retinite onchocerquienne. Bull Soc Pathol Exot 49:434, Favory A and Lagraulet J: Atrophie optique et lesions retiniennes dans l'onchocercose oculaire. Bull Mem Soc Fr Ophthalmol 69:393, Choyce DP: IV. Some observations on the ocular complications of onchocerciasis and their relationship to blindness. Trans R Soc Trop Med Hyg 52:112, D'Haussey RR, LeBretton Oliveau G, Aubry M, Vandendorpe A, Lagraulet J, Budden FH, Bisley GG, and Quarcoopone CO: Ocular lesions of onchocerciasis. Lancet ii:960, D'Haussy R [sic] and Uemura K: A propos de l'origine genetique des lesions chorioretiniennes observees chez les onchocerquiens. Maroc Med 39:460, Rodger FC: Acute ocular onchocerciasis and its treatment. Br J Ophthalmol 41:544, Rodger FC: Posterior degenerative lesion of onchocerciasis. Br J Ophthalmol 42:21, Quere MA, Basset A, Lariviere M, Basset M, and Razafinjato R: Etude statistique sur la frequence et la specificite des complications oculaires de l'onchocercose. Bull Soc Med Afr Noire Lang Franc 8:1, Lagraulet J: Epidemiology of ocular onchocerciasis in Frenchspeaking countries of West Africa. Isr J MedSci8:1153, Neumann E and Gunders AE: Pathogenesis of the posterior segment lesion of ocular onchocerciasis. Am J Ophthalmol 75:82, Greene BM, Taylor HR, Brown EJ, Humphrey RL, and Lawley TJ: Ocular and systemic complications of diethylcarbamazine therapy for onchocerciasis: Association with circulating immune complexes. J Infect Dis 147:890, Newsome DA, Hewitt AT, Quinn TC, Semba RD, Newland HS, Taylor HR, Greene BM: Immune status alteration in onchocerciasis, with and without retinopathy. Proceedings of the XXV International Congress of Ophthalmology, Rome, Italy, May 4-10, Chan CC, Nussenblatt RB, Kim MK, Palestine AG, Awadzi K, Ottesen EA: Immunopathology of ocular onchocerciasis: 2. Anti-retinal antibodies in serum and ocular fluids. Ophthalmology 94:439, Taylor HR, Murphy RP, Newland HS, White AT, D'Anna SA, Keyvan-Larijani E, Aziz MA, Cupp EW, and Greene BM: Treatment of onchocerciasis: The ocular effects of ivermectin and diethylcarbamazine. Arch Ophthalmol 104:863, John T, Barsky HJ, Donnelly JJ, Rockey JH: Retinal pigment epitheliopathy and neuroretinal degeneration in ascaridinfected eyes. Invest Ophthalmol Vis Sci 28:1583, 1987.