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 29:1642-1651,1988

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 onchocerciasis 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

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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.

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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 unsensitized 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 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 vitritis (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 vitritis was present with mild disc edema and venous engorgement. Loss of pigment was present in small focal areas and appeared as choroidal hyperfluores- Fig. 1. (A) Vasculitis and intraretinal hemorrhages in an eye of a cence with leakage on fluorescein angiogram (Fig. 2B, naive monkey which received ten micronlariae in the vitreous. (B) C). By day 7, the small areas which exhibited pigment Fluorescein angiogram of the same fundus demonstrating leakage loss became enlarged and confluent (Fig. 2D) and from terminal venules. continued to show choroidal hyperfluorescence and leakage (Fig. 2E). Intravitreal injection of 500 micronlariae in an unsensitized monkey eye resulted in a moderate vitritis, mild disc edema with venous en- Clinical slit-lamp examination, anterior segment gorgement, and small intraretinal hemorrhages tem- photography, fundus photography, and fluorescein poral to the macula. By day 7, large focal areas of angiography were performed 1 day prior to intravit- retinal pigment loss were noted temporal to the mac- real injection and on the third and seventh days fol- ula and showed marked leakage. lowing injection. Ocular inflammation was graded according to the scheme of Schlaegel.14 All monkeys Intravitreal injection of micronlariae in the sensi- were sacrificed on day 7 after intravitreal injection, tized monkeys resulted in an anterior uveitis which and the eyes were preserved in glutaraldehyde and ranged from a mild reaction in the eye which received processed for light and electron microscopy. The ten micronlariae to a small hypopyon in the eye study was conducted in accordance with the ARVO which received 500 micronlariae. In general, the an- Resolution on the Use of Animals in Research. terior uveitis was most pronounced on the first day after intravitreal injection and gradually resolved to- ward day 7. In the sensitized monkey, injection often Results micronlariae resulted in a mild vitritis, mild disc The inflammatory reaction in the two unsensitized edema with venous engorgement, and two small in- monkeys was overall more severe than that seen in traretinal hemorrhages in the superotemporal mid- the two sensitized monkeys. Both the degree and du- periphery. On day 3, fluorescein angiogram demon- ration of anterior uveitis and chorioretinitis was strated mild disc leakage which resolved by day 7. greater in unsensitized monkeys (Table 1). The control eye remained normal. Injection of 50 Sensitization of systemically immunized monkeys micronlariae in the sensitized monkey resulted in a was confirmed by detection of IgG antibody to mi- mild vitritis, mild disc edema and venous engorge- crofilarial antigens in serum by ELISA. Preimmuni- ment, and trace vascular sheathing on day 3. By day

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Fig. 2. (A) Vasculitis, intraretinal hemorrhage, and RPE changes at day 3 in an eye of a naive monkey which received 50 microftfar- iae. (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 Histologic studies of eyes from unsensitized mon- focal vitritis was noted temporal to the macula. In the keys which were injected with ten microfilariae other eye, the injection of 500 microfilariae caused a showed mild vasculitis with eosinophils and intrareti- moderate vitritis with two patchy areas of pigment nal hemorrhages, and the eyes which received 50 and loss temporal to the macula. By day 7, the areas of 500 microfilariae showed extensive intraretinal hem- pigment loss became large and confluent (Fig. 3); and orrhages, perivascular inflammatory cell infiltrates, on fluorescein angiogram, the area appeared as reti- and loss of pigment and vacuolation of the RPE (Fig. nal pigment epithelial (RPE) transmission defects 5). Retinal arteries in the midperipheral retina had with leakage (Fig. 4). Fluorescein angiogram on day 3 perivascular infiltrates of eosinophils (Fig. 6). Retinal showed small punctate areas of choroidal hyperfiuo- edema and perivascular infiltrates were present along rescence with leakage. These focal areas later ap- retinal venules. Mild vitreous hemorrhages with fi- peared as small RPE defects by day 7. brin and enmeshed eosinophils, neutrophils, and

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Fig. 3. Large area of pigment loss from the RPE in an eye of an Fig. 4. Fluorescein angiogram of the same fundus shown in Fig- immunized monkey which received 500 microfilariae in the vitre- ure 3., demonstrating RPE transmission defects with choroidal hyperfluorescence.

macrophages were present in these eyes. Focal cho- ina adjacent to the choroidal microgranulomas roidal inflammatory cell infiltrates consisting of eo- showed RPE hypertrophy and hyperplasia and patho- sinophils and plasma cells were present in a patchy logic alterations in the other retina (Fig. 7). Patho- distribution throughout the posterior segment. Ret- logic findings in the sensitized monkeys included in-

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Fig. 5. Pigment loss and duplication of the RPE in a naive monkey which received 50 intravitreal microfilariae (magnification X571).

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Fig. 6. Retinal artery in midperipheral retina showing perivascular infiltrate of eosinophils (magnification X578).

traretinal hemorrhages in the eye which received ten caused by a circulating toxin secreted by adult micronlariae, perivasculitis with eosinophils in the worms. In a review of the previous studies by Hissette eye which received 50 micronlariae (Fig. 8), and ex- and Bryant and an additional case report,18 Ridley19 tensive eosinophilic perivasculitis and choroiditis in concluded that dead microfilariae provoke an inflam- the eye which received 500 micronlariae. Micronlar- matory reaction with occlusion of smaller blood ves- iae were found in the retina which had no surround- sels. He suggested that perivascular infiltration and ing inflammatory infiltrates but were located adja- endothelial proliferation contributed to the chorioret- cent to areas of RPE hypertrophy and hyperplasia inal degeneration. It also has been speculated that (Fig. 9). deficiency of a B complex vitamin played an etiologi- cal role.20 The circulating toxin theory was further supported by Budden,21 who noted that the lesions Discussion did not correlate with the intensity of infection, and The presence of live Onchocerca micronlariae in by others based on histopathological study.22-23 the vitreous of the cynomologus monkey eye pro- Choyce24 believed that the fundus lesion was not re- duces chorioretinal lesions which resemble those seen lated to onchocerciasis at all but was rather a heredi- in human ocular onchocerciasis. This suggests that tary tapetoretinal degeneration, a widely disputed posterior pole lesions may be directly related to the view.1-25'26 presence of micronlariae. Several cases of florid posterior exudative uveitis Many theories for the pathogenesis of the posterior have been observed in onchocerciasis.27 The histopa- pole lesions of onchocerciasis have been advanced thology from enucleated eyes revealed retinal hemor- since they were first described by Hissette.1516 rhages and RPE degeneration, hypertrophy and hy- Bryant17 suggested that the fundus lesions were perplasia near microfilariae.27 Rodger believed that

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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 vasculitis.9-35 It is not known whether this low level presence of microfilariae and that the usual chorioret- inflammation is related to subsequent chorioretinal inal lesions associated with onchocerciasis were due changes, but both clinical and histopathological ob- to vitamin A deficiency combined with a circulating servation suggest that active infiltration of the retina toxin from adult worms.28 Subsequent studies have by microfilariae may chronically and progressively not shown any evidence that systemic vitamin A de- lead to widespread chorioretinal damage. The intra- ficiency plays any role in the disease.'•293° A histo- retinal hemorrhages, vasculitis and RPE loss seen in pathological study of the typical advanced posterior the present study resemble the onchocercal lesions pole lesion showed microfilarial debris within areas of seen in humans. The degree of inflammation in these posterior pole inflammation and live microfilariae in monkey eyes was generally greater than that seen in 31 the atrophied retina. There was no histologic evi- human onchocerciasis. A small hypopyon or fibrin dence of vascular occlusion to support the mecha- clot in the anterior segment is not typical for human 19 nism postulated earlier by Ridley. More recently, ocular onchocerciasis. The degree of retinal inflam- 32 Greene and coworkers have postulated that circu- mation observed clinically is greater in the animal lating immune complexes may play a role in the cho- model than in the human disease. It is possible that rioretinal changes of onchocerciasis. Antiretinal anti- O. lienalis, the species causing bovine onchocerciasis, bodies are present in patients with onchocercal cho- 3334 is more antigenic to primates than O. volvulus, thus rioretinitis, but the significance of circulating accounting for the greater severity of disease. A diffi- antibodies to retinal tissue is unclear. culty inherent in creating an animal model for Recent clinical observations of patients with on- human onchocercal chorioretinitis is that the human chocerciasis revealed live intraretinal microfilariae disease appears to be characterized by chronic, insidi- and intraretinal hemorrhages, cotton wool spots and ous chorioretinal damage with low-level inflamma-

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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 crofilariae in the eye. Chorioretinal damage may would be difficult to study an animal model which occur from host reaction to excretory-secretory anti- had the same slow progression. gens deposited by live microfilariae or a reaction to Our observations suggest that even small numbers dead or dying microfilariae. Eosinophil granule of live microfilariae in the vitreous can lead to chorio- major basic protein or eosinophil-derived neuroretinal damage. As few as ten microfilariae in the toxin, which may be toxic to the retina and choroid, vitreous were sufficient to cause chorioretinitis with could be released by accumulated eosinophils and retinal vasculitis, choroiditis and pathologic alter- elicit pathologic alterations.36 Inflammation of the ations in the RPE and outer retina. In this animal choroid with subsequent choroidal atrophy could model, chorioretinal damage appeared to occur both lead to outer ischemic atrophy of the retina. from inflammation associated with the retinal vascu- The differences in the ocular response to microfi- lature and inflammation associated with the chorio- lariae between the sensitized and unsensitized mon- capillaris. The main feature of human eyes affected keys were pronounced in that the unsensitized mon- by ocular onchocerciasis appears to be choroiditis keys developed a more severe reaction with a hemor- and atrophy of the choroid,831 and retinal vasculitis rhagic retinal vasculitis. This suggests that previous does not appear to be important in the pathogenesis exposure to systemic microfilariae may possibly of the posterior pole lesion. The retinal vasculitis seen downgrade the subsequent ocular response to micro- in the animal model may have been much more pro- filariae. nounced than in the human disease because live mi- Further studies are needed to determine the long- crofilariae were injected directly into the vitreous, term effect of intravitreal microfilariae on the retina, and the vitreous may not be the usual route for mi- choroid and optic nerve and to identify the immuno-

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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 pathogene- 5. Duke BOL and Garner A: Fundus lesions in the rabbit eye sis of the posterior segment changes. following inoculation of Onchocerca volvulus microfilariae into the posterior segment: 1. The clinical picture. Trop Med Key words: onchocerciasis, Onchocerca, chorioretinitis, Parasitol 27:3, 1976. animal model, microfilaria 6. 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, 1976. Acknowledgments 7. Donnelly JJ, Taylor HR, Young EM, Khatami M, LokJB, and The authors wish to acknowledge and thank Shirley Rockey JH: Experimental ocular onchocerciasis in cyno- Johnson and Sam D'Anna for technical assistance in the molgus monkeys. Invest Ophthalmol Vis Sci 27:492, 1986. project and Inga Jackman and Alice Flumbaum for help in- 8. Paul EV and Zimmerman LE: Some observations on the ocu- preparation of the manuscript. lar pathology of onchocerciasis. Hum Pathol 1:581, 1970. 9. Murphy RP, Taylor HR, and Greene BM: Chorioretinal damage in onchocerciasis. Am J Ophthalmol 98:519, 1984. References 10. Greene BM, Gbakima AA, Albiez EJ, and Taylor HR: Hu- moral and cellular immune responses to Onchocerca volvulus 1. Bird AC, Anderson J, and Fuglsang H: Morphology of poste- infections in humans. Rev Infect Dis 7:789, 1985. rior segment lesions of the eye in patients with onchocerciasis. 11. Greene BM, Fanning MM, and Ellner JJ: Non-specific sup- Br J Ophthalmol 60:2, 1976. pression of antigen-induced lymphocyte blastogenesis in On- 2. Taylor HR: Report of a workshop: research priorities for im- chocerca volvulus infection in man. Clin Exp Immunol 52:259, munologic aspects of onchocerciasis. J Infect Dis 152:389, 1983. 1985. 12. Bianco AE, Ham P, El Sinnary K, and Nelson GS: Large-scale 3. Donnelly JJ, Rockey JH, Taylor HR, and Soulsby EJL: On- recovery of Onchocerca microfilariae from naturally infected chocerciasis: experimental models of ocular disease. Rev Infect cattle and horses. Trans R Soc Trop Med Hyg 74:109, 1980. Dis 7:820, 1985. 13. Ham P, Towson S, James ER, and Bianco AE: An improved 4. Lagraulet MJ: Chorio-retinite onchocerquienne experimentale technique for cryopreservation of Onchocerca microfilariae. chez le lapin. Bull Soc Ophthalmol Fr 58:8, 1957. Parasitology 83:139, 1981.

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