experimental acanthamoeba keratitis: i. preliminary findings · uses of an animal model of...

BritishJournal ofOphthalmology, 1990,74,551-555

Experimental Acanthamoeba keratitis:I. Preliminary findings

D F P Larkin, D L Easty

AbstractExperimental Acanthamoeba keratitis wasinduced in Wistar rats by intrastromalinoculation ofAcanthamoebapolyphaga cysts.Keratitis developed in all corneas injected andwas characterised by a diffuse granularopacity. The animals were examined for 70days and a gradual reduction of keratitis wasobserved. Pathological sections showedliquefactive stromal necrosis andAcanthamoeba localised in deep stromapersisting to 70 days. A brisk inflammatoryinfiltrate of neutrophils and macrophages wasseen in the early days of infection. Severity ofkeratitis was found to correlate with inflam-matory activity in pathological sections. Theuses of an animal model of Acanthamoebakeratitis are described.

Department ofOphthalmology, BristolEye Hospital, BristolD F P LarkinD L EastyCorrespondence to:Mr D F P Larkin, MoorfieldsEye Hospital, City Road,London EC1V 2PD.Accepted for publication29 March 1990

Acanthamoeba is a genus of free-living protozoonwith a ubiquitous distribution in nature. It has alife cycle of active trophozoite and dormant cyststages. First isolated and described in 1913,' itwas not considered to have pathogenic potentialuntil reported to cause fatal meningoencephalitisin animal inoculation experiments in 1959.2Some years later, when suspected amoebicencephalitis was reported in humans, thesimilarity to Acanthamoeba infection inducedexperimentally was recognised and suggested theaetiological role of Acanthamoeba. The patho-logical features of human encephalitis are sosimilar to those of experimental Acanthamoebaencephalitis in mice that the use of animalmodels for studying other forms ofAcanthamoebainfection is clearly indicated.3

After initial reports45 Acanthamoeba infectionof the cornea remained a rare disease until anassociation with contact lens wear was firstrecognised.6 Reports of cases have exponentiallyincreased since that time, and a recent analysisidentified 85% of affected patients as contact lenswearers.7 Acanthamoeba keratitis typically has anindolent but progressive course, with intensediscomfort and stromal infiltration. Therapy ofAcanthamoeba keratitis is difficult in all cases,with frequent failures of medical and surgicaltreatment and loss of the eye in some of these.Even though the keratitis has become more

common than the encephalitis, few laboratoryin-vivo studies have been reported. Rabbitmodels have been developed, first by Font andcolleagues.8 9 After subconjunctival adminis-tration of corticosteroid for four days the directintrastromal injection of A polyphaga inducedstromal keratitis and corneal ulceration. Somecorneas were culture-positive at intervals, andencysted amoebae were observed in tissue on

microscopy. A further rabbit model was reported

by Lin and colleagues in which the effect ofsteroid administration was analysed.'0 Badenochet al reported a model in Porton rats in whichinduction of Acanthamoeba keratitis wasdependent on coinoculation with bacteria. II

In this study the Wistar rat was used as theexperimental animal. The initial objective of thestudy was to induce reproducible disease similarto the human disease, for use in evaluation ofimmunopathology and therapeutic agents. Sothat the specific immune response to Acanth-amoeba could be studied coinoculation withbacteria was avoided.

Materials and methodsAnimals. Male Wistar rats weighing approxi-mately 125 g were used. The corneas wereexamined prior to inoculation to excludeabnormality (Fig IA). There were 24 rats in theinfected group and six in the control group. Allprocedures were performed on the right eye.Amoeba. All studies were performed with the

Shinh strain of A polyphaga, a human cornealisolate (kindly provided by Mr S Kilvington,Public Health Laboratory, Bath). This had beenfrozen in liquid nitrogen and passaged on a Verocell monolayer three times. An axenic suspensionof Ix 106 amoebae/ml (90% cysts) was preparedin Page's amoeba saline. 12

Anaesthesia. Diazepam 0 25 mg and fentanyl0.01 mg/fluanisone 0 3 mg were given by intram-uscular injection, and one drop of preservative-free benoxinate was applied to the right eye.

Inoculation technique. The procedure wasperformed under an operating microscope(OPMI 1 FC, Carl Zeiss Ltd). Initially a halfthickness linear blade incision was madeapproximately 2 mm from the centre of thecornea. With a microlitre syringe (HamiltonBonaduz AG) and a sterile 30 G needle (Becton-Dickinson & Co.), the needle was advanced fromthe incision through the lamellae of the stromato the centre of the cornea. 1 [1 was injectedinto the stroma. Control animals received amock inoculum of 1 [1 of Page's saline; infectionanimals received a suspension of 1 x 106 amoebae/ml (90% cysts) in Page's amoeba saline.

Clinical evaluation Under fentanyl generalanaesthesia the rats were examined with a slit-lamp microscope on days 1, 3, 5 and 7 followinginoculation and weekly therafter until 70 days.Examination was by retroillumination, resultingin transillumination of the albino iris and thecornea. The oblique slit beam was used toilluminate lesser degrees of opacity. A clinicalgrading system was used to describe cornealopacity:Grade 3: opacity visible on retroillumination,

obscuring iris vessel detail.

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Grade 2: opacity visible on retroillumination,but not sufficient to obscure iris vessels.Grade 1: opacity visible only by oblique slit

beam.Grade 0: normal.A mean opacity grade was calculated for the

infected corneas on each examination day.Photographs were taken with the operatingmicroscope with retroillumination of the cornea,160 ASA tungsten film (Kodak Ektachrome) andautomatic exposure.On days 1, 3, 7, 14, 21, 28, 49, and 70 after

inoculation two infected rats were killed byintraperitoneal injection of 60 mg pentobarbi-tone. Two control rats were killed on days 1, 3,and 7.

Tissue processing. The eye was enucleated andthe cornea dissected from the globe andhemisected. One half of the cornea was fixed for24 h in neutral buffered formaldehyde, embed-ded in wax, and 6 [tm thickness sections cut witha microtome; the other half was either immedi-ately frozen in liquid nitrogen or homogenisedwith a glass grinder for culture.

Preparation of specific antibody. Rabbitantiserum was prepared to the experimentalstrain of A polyphaga according to the methoddescribed by Warhurst.'3 Briefly, a 2-5 kg NewZealand White rabbit was injected intramus-cularly with 30 ml A polyphaga/Freund'scomplete adjuvant and this was repeated at athree-week interval. This was followed by two0-6 ml intravenous injections of formalinised Apolyphaga cysts at four and five weeks. Aftervenepuncture at six weeks the antiserum titrewas measured and found to be 1/1024. Theserum was then affinity purified on a chroma-tography column using protein A-Sepharose(Sepharose CL-4B, Pharmacia, Uppsala,Sweden) to yield the IgG fraction. 14

Staining ofwax sections. Sections were stainedwith haematoxylin and eosin or with rabbit anti-Acanthamoeba antibody. For the latter proceduresections were first incubated at room tempera-ture with 1:5 normal swine serum (Dakopattsa/s) to block non-specific background staining;they were then incubated in sequence with rabbitanti-Acanthamoeba antibody at dilution 1:10 for30 minutes, and peroxidase-conjugated swineanti-rabbit immunoglobulin (Dakopatts a/s) atdilution 1:50 for 30 minutes. Phosphate-bufferedsaline (PBS) was used for all dilutions, andsections were washed with PBS following eachof these incubations. 3,3'-Diaminobenzidine(Sigma Chemical Co.) was the chromagen usedto identify antibody binding, mixed with H202in Od M trometamol (Tris) buffer, giving abrown end-product: sections were incubated forthree minutes and then washed in tap water forfive minutes. Sections were counterstained withhaematoxylin.

Culture of corneas for Acanthamoeba. Non-nutrient agar was set in each well of 25-well Petriplates. A lawn of live Escherichia coli was added

Figure IA Normal ratcornea withtransillumination ofalbinoiris seen on retroillumination.

Figure IB Day 5: infectedrat cornea showing diffusegranular central cornealopacity.

Figure IC Day 14:clearance ofmuch ofthecorneal oedema andmaximal opacity at theinoculation site (arrow).

Figure ID Day 35:paracentral ring-shapedgranular opacity andfinesuperficial peripheralvascularisation (arrow),photographed followingmydriatic administration.

to the agar surface in each well, and after 8 hours'incubation at 37°C the plates were ready forinoculation with tissue. Hemicorneas werehomogenised in 150 ii of Page's saline with aglass grinder, 10 [d of the undiluted homogenateadded to each well, and the plate sealed. Corneas

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Experimental Acanthamoeba keratitis in the Wistar rat: I. Preliminaryfindings

from control and infected eyes were cultured andobserved by microscopy for seven days. If nogrowth was seen by seven days, the culture wasdeemed negative.

Results

CLINICAL EVALUATIONThe cloudy opacification observed immediatelyafter injection of all corneas cleared completelyin control rats before the first examinationday 1. Reepithelialisation of the linear incisionhad also occurred by this time.

All rats inoculated with Acanthamoebadeveloped keratitis. Certain clinical featureswere consistently observed. Opacitywas granularin character, with corneal thinning superficial toinfiltrates apparent in animals with more long-standing keratitis. Minimal, if any, vascular-isation and no corneal perforation was seen (FigsiB, IC, 1D). No epithelial disease and no signsof anterior uveitis such as nodular infiltrates orhypopyon were observed.On days 1, 3, and 5 following inoculation

grade 3 opacity was observed in almost all rats,maximal at the inoculation site. On day 7 cornealopacity was grade 3 in most rats but haddiminished to grade 2 in some. On subsequentexaminations a gradual reduction in mean gradeof corneal opacity was found (Table I).

EXAMINATION OF PATHOLOGICAL SECTIONSHaematoxylin and eosin (H-E) staining ofinfected corneas identified a marked inflam-matory response ofneutrophils and macrophagesat the inoculation site on day 1 (Fig 2A). Adistinction between Acanthamoeba and inflam-matory cells was not possible by H-E stain butwas facilitated by immunoperoxidase stainingwith anti-Acanthamoeba antibody (Fig 2B).Excystment of many amoebae (Fig 2C) and

Figure 2A Day 1: non-specific staining byamoebae and inflammatorycells at the inoculation sitein an infected cornea. (H-E,x540.) They are very

difficult to distinguish.

P.

Figure 2B Day 1: Acanthamoeba cysts (large arrow) andmigrating polymorphs (small arrow). Little excystment hastaken place. (Anti- Acanthamoeba antibody x 300).

trophozoite migration from the inoculation sitewas seen by day 3. A more subdued and mixedinflammatory response was evident by this time,with lymphocytes and monocytes appearingamongthe neutrophils and macrophages seen on day1. By day 7 trophozoites were seen throughoutthe cornea, though large numbers of cystsremained at the inoculation site. Marked stromalthinning was seen overlying the inoculation sitein some corneas at day 14 (Fig 2E). Examinationof corneas late in disease, from days 21 to 70,consistently revealed amoebae and inflammatorycells to be localised to the inoculation site,surrounded by eosinophilic material.A gradually diminishing inflammatory

response was seen in control corneas at 1, 3 and 7days after mock inoculation (Fig 2F).

CULTURE OF CORNEAPositive Acanthamoeba cultures resulted frominfected corneas of all animals killed at days 1-49inclusive. The homogenised corneas of the ratkilled at day 70 and all control rats were culture-negative.

DiscussionThe value of an animal model of Acanthamoebakeratitis is attested to by the gravity andincreasing prevalence of clinical disease inhumans. Study of Acanthamoeba keratitis in ananimal model allows control of many of thevariable factors involved in human disease. In

Table I Mean grade ofkeratitis observedfollowing injectionofAcanthamoeba

Day Number ofrats Mean grade ofexamined keratitis (SD)

1 24 2-72 (0-60)3 22 2-76(0-61)5 20 2-77(0-59)7 .18 2-42 (0-79)

10 16 2-08(0-81)14 14 1-75 (0-65)21 12 1-71(0-76)28 10 1-93 (0-83)42 8 1-29(0-49)70 6 1-50(0 58)

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*4.: m............-~.....; .A.* X.Figure 2C Day 1:excysting Acanthamoeba(thin arrow) at the site ofthe linear blade incision,indicated by epithelialdowngrowth (wide arrow).Anti-Acanthamoebaantibody, x300.)

Figure 2D Day 7: cyst(large arrow) andtrophozite (small arrow),each being killed by severalinflammatory cells (Anti-Acanthamoeba antibody,x 760.)

Figure 2E Day 14:marked thinning ofcornealstroma overlyingAcanthamoeba cysts whichare in deep stroma. Thesehave not migrated and rest onDescemet's membrane.(Anti-Acanthamoebaantibody, x 140.)

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particular, the clinical features of establishedstromal infection may be analysed, pathologicalstudy oftissue undertaken, and, in an establishedmodel, treatments evaluated. The most appro-

priate treatment for Acanthamoeba keratitis hasyet to be decided: insufficient correlationbetween in-vitro drug sensitivity tests andclinical response renders it difficult to evaluatenew drugs.

In this study Acanthamoeba keratitis wasinduced by inoculation oforganisms directly into

Figure 2F Day 3: control cornea showing low-gradeinflammation at inoculation site (arrow). (Anti-Acanthamoeba antibody, x 190.)

the corneal stroma. Therefore this model doesnot address questions on the means by whichinfection arises, but what follows once a largenumber of cysts reach the stroma. Reproduciblekeratitis developed in all rat corneas inoculatedwith 1 [d of a suspension of Acanthamoeba at aconcentration of 1 x 106/ml. Because in -clinicalpractice such an inoculum of cysts would be veryunlikely, this may indicate that Acanthamoebaare less infective for the rat cornea than thehuman. Certain clinical features mimic thoseobserved in human infection, such as minimalcorneal vascularisation and diffuse granularopacity formation rather than formation of anabscess. Although acute fulminating infectionwas not induced, the clinical course of theexperimental keratitis is shorter than the humandisease, which may remain active for severalmonths. This might be due to differences in ratand human susceptibility to infection. In general,clinical inflammation correlated with histopatho-logical indices of inflammation rather thanpresence ofamoebae as such. In accordance withthe report of Font et al.9 cysts seemed moreresistant to destruction than trophozoites.Most human specimens on which histopatho-

logical descriptions are available originate fromcorneal transplantation and accordingly describelate stages of the disease, probably modified bydrug therapy. Nevertheless, available reportsdescribe changes similar to those described inthis report. Disruption and necrosis of stromallamellae and invasion by amoebae ofdeep stromato Descemet's membrane are observed withinfiltrates of neutrophils; in some cases there isan added lymphocyte and plasma cell com-ponent.6 15-17 However, Acanthamoeba can beseen in tissue relatively free of inflammation. 7 8

Initial conclusions from this study indicatethat reproducible keratitis of short clinicalduration can be induced by intrastromal injectionof axenic Acanthamoeba cysts without the needfor concomitant immunosuppression bycorticosteroid8-'0 or coinoculation with bacteria."Thus the rat provides a useful model for inves-tigating the pathology ofAcanthamoeba keratitis.Further sequential studies are in progress to

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ExperimentalAcanthamoeba keratitis in the Wistar rat: I. Preliminaryfindings

characterise the cellular composition of theimmune response to Acanthamoeba. This modelwill provide improved understanding ofAcanthamoeba-host interactions and ultimatelywill allow evaluation of specific antimicrobialdrugs with and without corticosteroid.

This study was supported in part by grants from Alcon LaboratoriesLtd, the British Council for the Prevention of Blindness, and theSouth-Western Regional Health Authority.The authors are indebted to Simon Kilvington, BSc, for initially

providing Acanthamoeba cysts and assisting with preparation ananti-Acanthamoeba antibody; and to Ian Williams for assistancewith the preparation of tissue sections.

1 Puschkarew BM. OYber die Verbreitung der Susswasser-protozoen durch die Luft. Arch Protistenk 1913; 28: 323-62.

2 Culbertson CG, Smith JW, Cohen HK, Minner JR. Experi-mental infection ofmice and monkeys byAcanthamoeba. AmJPathol 1959; 35: 185-97.

3 Martinez AJ. Free-LivingAmebas: Natural History, Prevention,Diagnosis, Pathology, and Treatment ofDisease. Boca Raton:CRC Press, 1985; 127-44.

4 Jones DB, Visvesvara GS, Robinson NM. Acanthamoebapolyphaga keratitis and Acanthamoeba uveitis associated withfatal meningoencephalitis. Trans Ophthalmol Soc UK 1975;95:221-32.

5 Nagington J, Watson PG, Playfair TJ, McGill J, Jones BR,Steele ADMcG. Amoebic infection of the eye. Lancet 1974;ii: 1537-40.

6 Moore MB, McCulley JP, Luckenbach M, et al. Acanthamoebakeratitis associated with soft contact lenses. AmJ Ophthalmol1985; 100:396-403.

7 Stehr-Green JK, Bailey TM, Visvesvara GS. The

epidemiology ofAcanthamoeba keratitis in the United States.AmJOphthalmol 1989; 107:331-6.

8 Font RL, Tapert MJ, Robinson NM, Osato MS, Jones DB.An animal model ofAcanthamoeba keratitis. Invest Ophthal-mol Vis Sci 1981; 20 supply): 8.

9 Font RL, Tapert MJ, RobinsonNM, et al. An animal model ofAcanthamoeba keratitis. Further studies with emphasis onthe early phase destruction of the trophozoites. InvestOphthalmol VisSci 1982; 22 (suppl): 163.

10 Lin J, John T, Sahm D, Rockey JH. Animal model ofAcanthamoeba keratitis: effect of corticosteroids. InvestOphthalmol VisSci 1989; 30: (suppl): 37.

11 Badenoch PR, Johnson AM, Christy PE, Coster DJ. Patho-genicity of Acanthamoeba and a corynebacterium in the ratcornea. Arch Ophthalmol 1990; 108: 107-12.

12 Page FC. Taxonomic criteria for limax amoebae, with descrip-tions of 3 new species ofHartmannella and 3 of Vahlkamphia.J Protozool 1967; 14: 499-521.

13 Warhurst DC. Naegleria and Acanthamoeba in tissue sections.In: Edwards JMB, Taylor CED, Tomlinson AH, eds.Immunofluorescence techniques in diagnostic microbiology.London: HMSO, 1982; 46-8.

14 Tucker DF, Begent RHJ, Hogg NM. Characterization ofimmunecomplexes in serum by adsorption on staphylococcalprotein A: model studies and application to sera of ratsbearing a Gross virus-induced lymphoma. J Immunol 1978;121: 1644-51.

15 Lund OE, Stefani FH, Dechant W. Amoebic keratitis: aclinicopathological case report. Br J Ophthalmol 1978; 62:373-5.

16 Auran JD, Starr MB, Jakobiec FA, Acanthamoeba keratitis. Areview of the literature. Cornea 1987; 6: 2-26.

17 Garner A. Pathology of corneal acanthamoebic infection.In: Cavanagh HD, ed. The cornea: transactions of the WorldCongress on the Cornea III. New York: Raven Press, 1988:535-9.

18 Blackman HJ, Rao NA, Lemp MA, Visvesvara GS.Acanthamoeba keratitis successfully treated with penetratingkeratoplasty: suggested immunogenic mechanisms ofaction.Cornea 1984:3:125-30.

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