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Experimental Staphylococcic Keratitis

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Experimental Staphylococcic Keratitis Experimental staphylococcic keratitis Michael J. Hogan, Victor Diaz-Bonnet, Masao Okumoto, and Samuel J. Kimura The human clinical manifestations of staphylococcic infections of the eyelids, conjunctiva, and cornea are discussed. The corneal lesions occurring during the course of staphylococcic in- fections of the lid develop after the disease has been present in the eyelid for several years, but are not always correlated with the degree of activity. The peripheral infiltrations and ulcerations either are due to hypersensitivity or are a result of some product produced by Staphylococcus aureus. Repeated instillations of staphylococcic antigen and toxin onto the corneas of nonimmunized rabbits failed to produce the superficial epithelial keratitis which is so common in human beings with chronic staphylococcal infections of the lid. Injection of a small challenging dose of staphylococcic antigen into the center of the corneas of rabbits, which had been immunized over a period of time with subcutaneous staphylococcic antigen or toxoid, produced corneal rings resembling those in antigen-antibody reactions of the cornea. The incidence of rings was twice as common in those animals immunized with killed and dis- rupted staphylococci as in those immunized with toxoid. The ring infiltrations which were produced appeared within 24 to 48 hours, were complete or incomplete, lasted 3 to 5 days, and gradually disappeared without scarring. A second ring developed in a few animals. Intra- corneal injection of similar doses of toxoid into another series of similarly immunized animals failed to produce ring infiltrations. T ment of the lids in other cutaneous dis- _Lhe staphylococcus is one of the most eases, such as acne rosacea, is frequently important eye pathogens. Lesions produced associated with secondary staphylococcic by this organism on the lid, conjunctiva, infections. Most often, however, the lid and cornea have been studied for many margins are affected primarily by a staphy- years. lococcic infection which produces bleph- Cutaneous staphylococcic infections af- aritis. Seborrheic blepharitis is another fecting the lids, such as the pyodermas common disease of the eyelid which often which produce folliculitis, furuncles, and becomes secondarily infected with patho- cellulifis often are associated with second- genic staphylococci. The infection in bleph- ary conjunctivitis and keratitis. Involve- aritis may extend to the conjunctival sac to produce a conjunctivitis which is either acute or chronic. Alterations of the con- From the Department of Ophthalmology and the Francis I. Proctor Foundation for Research in junctival inflammation may or may not oc- Ophthalmology, University of California School cur with exacerbations and remissions of of Medicine, San Francisco, Calif. the inflammation of the lid. Since staphy- This investigation was supported in part by the lococci are not epithelial parasites, it has National Council to Combat Blindness Grant- been postulated that they produce kerato- in-Aid No. G-238, and by a Special Fellowship, conjunctivitis through the action of potent BT-666, from The National Institute of Neuro- exotoxins.1 Allen2 showed experimentally logical Diseases and Blindness, United States Public Health Service, Bethesda, Md. that Staphylococcus aureus toxin, when 267 Downloaded from jov.arvojournals.org on 09/28/2021 268 Hogan et al. Investigative Ophthalmology April 1962 dropped into the eye, can result in super- time to time, especially in the chronic vari- ficial punctate erosions of the corneal epi- eties of conjunctivitis. It tends to be more thelium. pronounced on arising in the morning, Except for fine diffuse punctate super- when the symptoms also are worse. During ficial epithelial erosions which stain with the day the erosions heal and symptoms fluorescein, the human cornea rarely is af- diminish. Scrapings of the lid margin fected during all these staphylococcic in- usually show an abundance of gram-posi- fections. Occasionally, during the course of tive cocci, and cultures verify the presence some infections of the lid however, mar- of pathogenic Staphylococcus aureus. Con- ginal corneal infiltrations and ulcerations junctival scrapings show epithelial cells, occur. Most often cultures from the ulcers lymphocytes, polymorphonuclear leuko- show no growth of organisms. It has been cytes, and, at times, staphylococci. postulated that these peripheral corneal Peripheral marginal infiltrations and lesions are due to the effects of staphylo- ulcerations also may appear during the coccic toxin, either as a direct toxic effect, quiet or active phase of the blepharitis. 3 J or to hypersensitivity. Woods and Burky Thygeson7 found Staphylococcus aureus on demonstrated that hypersensitivity to the lids in 133 of 156 catarrhal ulcers as- staphylococcic toxin may produce conjunc- sociated with chronic catarrhal con- tivitis. Very little, however, has been done junctivitis, and indicated the importance to explain the pathogenesis of the corneal of conjunctivitis in the production of the lesions in human beings. Germuth and co- corneal lesions. Single or multiple gray, 5 workers showed that an opaque ring could rounded, or crescentic lesions may develop be produced in the peripheral cornea of the in the peripheral corneal stroma, separated rabbit with bovine serum globulin and from the limbus by a narrow rim of normal albumin. This demonstrates that immuno- corneal tissue (Fig. 1). Any portion of the logic phenomena can be produced in the peripheral cornea may be affected, and at corneal stroma of experimental animals. times the multiple lesions extend to form 0 The Wessely phenomenon in experi- partial ring infiltrates. Only occasionally a mental animals also provides evidence that complete ring infiltration occurs around the hypersensitivity plays a part in the de- corneal periphery.7 The infiltration is 1 to velopment of experimental infiltrations of 2 mm. in breadth, and rarely extends the peripheral cornea. deeper than the midstroma. Ulceration is This study is one of a series designed to common (catarrhal ulcer). Recurrences are develop an experimental model in rabbits common over a period of years. Vasculari- to gain further information on the patho- zation occurs from the limbus. Cultures genesis of staphylococcic keratitis. from the ulcers are negative. Cytologic studies show acute inflammatory cells and Clinical manifestations necrotic corneal debris. There is a rapid It is known that corneal lesions are un- response of the lesions to corticosteroid common in children. It also is known that therapy. This suggests either that the in cases of staphylococcic blepharitis the lid lesions are due to some type of antigen- inflammation may be mild at the time the antibody reaction or that they are toxic and keratitis develops. are benefited by the anti-inflammatory The epithelial keratitis most often pro- corticosteroid effect. duces mild symptoms, such as smarting or Similar corneal ulcerations have been re- burning. It is characterized by punctate ported rarely with Moraxella lacunata and staining, which involves the lower half of by Hemophilus aegyptius, but in these con- the cornea, but occasionally it is more ditions the organism can be recovered from diffuse, even involving the upper limbal the conjunctiva. area. The staining varies in intensity from Thygeson7 reported 14 cases of ring in- Downloaded from jov.arvojournals.org on 09/28/2021 Volume 1 Experimental staphylococcic keratitis 269 Number 2 Fig, 1. Marginal infiltrate secondary to staphylococcus. filtration and ulceration of the cornea in sterile. It was placed in a sterile container and his series. The flora of the conjunctiva and refrigerated at 4° C. Toxoid. A commercial preparation of toxoid * lid margin was normal in 12 cases, and in 2 containing 10,000 units per milliliter was used. a coagulase-positive staphylococcus was Toxin. Crude staphylococcus toxinf was used found. It is known that similar lesions can (Lot No. 42925-199), and was preserved with also occur during the course of bacillary thimerosal 0.01 per cent. dysentery, periarteritis, and ulcerative Rabbits. Black Dutch rabbits of medium size colitis. were purchased from various animal suppliers. The eyes were examined with a slit lamp prior to carrying out the experimental work. All had Materials normal corneas. Antigen. A strain of mannitol- and coagulase- positive, hemolytic Staphylococcus aureus, isolated Methods from the lid of a patient with an external hordeolom was used. The organism was inocu- Immunization. Immunization with Staphylococ- lated on nutrient agar, incubated for 24 hours at cus aureus was carried out as follows: A partially 37° C., and harvested by scraping the colonies heat-killed suspension was mixed with an equal and suspending the organisms in saline solution. volume of Freund's complete adjuvant, f One milli- The resulting suspension showed a plate count of liter of this mixture was given subcutaneously 1010 live organisms per milliliter. A portion of each week for a total of 6 weeks. Agglutinin titers this antigen was heated for 2 hours at 60° C. for were determined at the end of 6 weeks, and were immunization of some rabbits. It was found that positive in all animals at a titer of 1:32 or more. heating the suspension for 2 hours at 60° C. failed Immunization of another series of rabbits was to kill all the organisms. Therefore, the freshly carried out by giving 1.0 ml. of toxoid and prepared suspension was placed in a sonic oscil- Freund's adjuvant subcutaneously each week for lator* for 2 hours at 0° to -3° C. The frequency 6 weeks. The presence of precipitin antibody to was 10 kilocycles per second at 250 watts. This toxin was demonstrated by use of the agar dif- failed to kill all organisms. Therefore, the sus- fusion technique. The results showed strongly pension was centrifuged at 1,000 r.p.m. for 10 positive bands with all sera. minutes. The supernatant fluid was then passed Challenge. The immunized and normal control through a millipore filter (H.A. 0.47 micron pore rabbits were challenged either by local instillation size). The filtrate was cultured and found to be *Lederle Laboratories, Penr] River, N.
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