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 cyno- molgus 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 29:1642-1651,1988 Onchocerciasis, a filarial infection caused by On- Experimental animal models have provided addi- chocerca volvulus, is a major cause of blindness in tional information on the immunopathology and endemic areas of Africa and Central and South pathogenesis of ocular onchocerciasis.3 Chorioretinal America. Much of the blindness from onchocerciasis lesions have been produced by intraorbital implanta- is due to a characteristic, progressive chorioretinal tion of O. volvulus adult worms4 and intravitreal and scarring,1 but the pathogenesis of this fundus lesion is subretinal injections of O. volvulus microfilariae in unclear.2 It is difficult to obtain affected human eyes the rabbit.5'6 Because O. volvulus microfilariae can for histopathological study, and the few eyes which only be obtained from humans and are difficult to have been studied exhibit far advanced disease with obtain in large quantity, the alternative use of On- other accompanying pathology. Thus, the pathogene- chocerca spp. microfilariae from other animals, such sis of the extensive chorioretinal damage in oncho- as O. cervicalis from the horse and O. gutturosa and cerciasis cannot be easily determined from human O. lienalis from cattle, has been explored.3 The use of material. closely related species of microfilariae appears to be a good alternative in animal models. The nonhuman primate eye is a more accurate an- From The International Center for Epidemiologic and Preven- atomical model than the lagomorph or rodent eye for tive Ophthalmology, The Wilmer Institute and the School of Hy- giene and Public Health, The Johns Hopkins University, Balti- producing lesions resembling human onchocerciasis. more, Maryland, the fDepartment of Ophthalmology, Scheie Eye In a recent study of experimental ocular onchocer- Institute, University of Pennsylvania School of Medicine, and the ciasis in cynomolgus monkeys, intravitreal injections ^Department of Pathobiology, University of Pennsylvania School of approximately 10,000 O. lienalis microfilariae led of Veterinary Medicine, Philadelphia, Pennsylvania. to an intense posterior and anterior chamber reaction Supported in part by NIH Grant EY-03324 (HRT), EY-00286 which precluded a view of the fundus.7 (RDS), EY-03984 (JHR), EY-06616 (JJD); and AI19995 (JBL) and by grants from the World Health Organization Special Program for The purpose of the present study is to determine Research and Training in Tropical Diseases (RDS) and Onchocer- whether the posterior segment lesions of onchocer- ciasis Control Programme Onchocerciasis Chemotherapy Project ciasis can be reproduced in cynomolgus monkeys by (JJD). intravitreal injection of small numbers of microfilar- Submitted for publication: March 5, 1987; accepted June 13, iae. This would more closely resemble the clinical 1988. 8 Reprint requests: Dr. Hugh R. Taylor, The Wilmer Institute, The situation, since both histopathological studies and 9 Johns Hopkins Hospital, Baltimore, MD 21205. clinical observations have shown that microfilariae 1642 Downloaded from iovs.arvojournals.org on 09/26/2021 No. 11 EXPERIMENTAL ONCHOCERCAL CHORIORETINITIS / Sembo er ol 1643 Table 1. Clinical findings in cynomolgus monkeys receiving intravitreal O. lienalis microfilariae Clinical status 1 week after inoculation Intravitreal Monkey Sensitized* Microfilariae] Anterior chamber Vitreous Retina/choroid 1 yes 500 O.D. Small hypopon Moderate Large patchy RPE vitritis pigment loss, leakage on fluorescein 50 O.S. Small fibrin clot Moderate Trace vascular vitritis sheathing and mild disc edema 2 yes 10 O.D. Mild anterior Trace Small intraretinal uveitis vitritis hemorrhages and trace disc edema OO.S. Normal Normal Normal (balanced salt solution) 3 no 500 O.D. Hyphema, fibrin Moderate Large patchy RPE clot vitritis pigment loss, small intraretinal hemorrhages, mild disc edema 50 O.S. Small fibrin clot Mild Large intraretinal vitritis hemorrhages, vasculitis, patchy RPE pigment loss, mild disc edema 4 no 10 O.D. Mild anterior Mild Moderate uveitis vitritis hemorrhagic vasculitis with sheathing OO.S. Normal Normal Normal (balanced salt solution) * 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, ret- Two of four cynomolgus monkeys (Macaca fascic- ina, and choroid, in spite of the extensive chorioreti- ularis) were sensitized with one subcutaneous injec- nal damage which has been observed. In addition, in tion of 23,000 O. lienalis microfilariae followed by vitro studies have suggested that onchocercal antigens another subcutaneous injection of 30,000 microfilar- may suppress the cellular immune response to infec- iae 1 week later. Each inoculum was distributed on tion by Onchocerca,7-1011 and thus the role of sensiti- four sites on the abdomen and given under ketamine zation on the subsequent ocular response was inves- anesthesia. Peripheral blood mononuclear leukocyte tigated. This study suggests that chorioretinal lesions (PBML) proliferative responses to O. volvulus adult resembling those of ocular onchocerciasis may be worm crude extract antigen have been demonstrated produced by small numbers of microfilariae. to increase five- to eight-fold following sensitization with similar doses of O. lienalis in other experiments Materials and Methods (unpublished). After sensitization, IgG antibody titers to microfilarial extract antigen were determined by Microfilariae of O. lienalis were obtained from the enzyme-linked immunoabsorbent assay (ELISA) as umbilical skin of cattle by the method of Bianco12 previously described.7 One week following the second and were cryopreserved using ethanediol.13 Motility inoculation, the sensitized and unsensitized monkeys of cryopreserved microfilariae was noted to be 90% or received an intravitreal injection of either 0 (control greater. Cryopreserved microfilariae were prepared injection), 10, 50, or 500 microfilariae in balanced for injection by thawing in Tyrode's solution with salt solution through a pars plana approach (Table 1). 20% fetal bovine serum (FBS), penicillin (100 units/ A 1 ml tuberculin syringe with a 30 gauge needle was ml), and streptomycin (100 fig/ml) warmed to 38°C; used to inject the 0.1 ml volume which contained washing three times in cold Tyrode's solution with microfilariae. Anterior chamber paracentesis was antibiotics but without FBS; and then being used im- performed after the intravitreal injections to relieve mediately for injection. intraocular pressure. Downloaded from iovs.arvojournals.org on 09/26/2021 1644 INVESTIGATIVE OPHTHALMOLOGY b VISUAL SCIENCE / November 1988 Vol. 29 zation ELISA absorptivities were 0.044 and 0.032, while postimmunization absorptivities were 0.299 and 0.395 in animals 1 and 2, respectively. Unsensi- tized monkeys had absorptivities of 0.047 and 0.074 (animals 3 and 4) at the time of intravitreal injection of micronlariae. Intravitreal injection of micronlariae in unsensi- tized monkeys resulted in a marked anterior uveitis which varied from a fibrin clot with keratoprecipi- tates in the eye which received 50 micronlariae to a more severe reaction which included a small hy- phema in the eye which received 500 micronlariae. Posterior segment changes in the eye of the unsensi- tized monkey which received ten intravitreal micro- nlariae included mild disc edema with venous en- gorgement and a severe retinal vasculitis with sheath- ing, adjacent intraretinal hemorrhages, but only minimal vitritis (Fig. 1 A). Fluorescein angiogram re- vealed 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).