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Corneal infection with Pseudomonas aeruginosa perforates the cornea in susceptible C57BL/6 (B6), but not resistant BALB/c, mice. To determine whether vasoactive intestinal peptide (VIP) played a role in development of the resistant response, protein expression levels were tested by immunocytochemistry and enzyme immunoassay in BALB/c and B6 corneas. Both mouse strains showed constitutive expression of corneal VIP protein and nerve fiber distribution. However, disparate expression patterns were detected in the cornea after infection. VIP protein was elevated significantly in BALB/c over B6 mice at 5 and 7 days postinfection. Therefore, B6 mice were injected with rVIP and subsequently demonstrated decreased corneal opacity and resistance to corneal perforation compared with PBS controls. rVIP- vs PBS-treated B6 mice also demonstrated down-regulation of corneal mRNA and/or protein levels for proinflammatory cytokines/chemokines: IFN-gamma, IL-1beta, MIP-2, and TNF-alpha, whereas anti-inflammatory mediators, IL-10 and TGF-beta1, were up-regulated. Treatment with rVIP decreased NO levels and polymorphonuclear neutrophil (PMN) number. To further define the role of VIP, peritoneal macrophages (Mphi) and PMN from BALB/c and B6 mice were stimulated with LPS and treated with rVIP. Treatment of LPS-stimulated Mphi from both mouse strains resulted in decreased IL-1beta and MIP-2 protein levels; PMN responded similarly. Both cell types also displayed a strain-dependent differential response to rVIP, whereby B6 Mphi/PMN responded only to a higher concentration of VIP compared with cells from BALB/c mice. These data provide evidence that neuroimmune regulation of the cytokine network and host inflammatory cells functions to promote resistance against P. aeruginosa corneal infection.

The role of macrophage inflammatory protein-1alpha (MIP-1alpha) in cell infiltration into Pseudomonas aeruginosa-infected cornea and subsequent disease was examined. Greater amounts of the chemokine (protein and mRNA) were found in the infected cornea of susceptible B6 ("cornea perforates") versus resistant BALB/c ("cornea heals") mice from 1 to 5 days postinfection. Treatment of BALB/c mice with recombinant (r) MIP-1alpha exacerbated disease and was associated with an increased number of neutrophils (PMNs) in the cornea. Treatment of BALB/c mice with rMIP-1alpha also induced recruitment of activated CD4+ T cells into the affected cornea, converting resistant to susceptible mice. Depleting CD4+ T cells in r-treated BALB/c mice significantly decreased PMNs in cornea tissue, suggesting that T cells regulate persistence of PMNs at this site. In B6 mice, administration of neutralizing MIP-1alpha polyclonal antibody also significantly reduced PMN numbers and pathology. Collectively, evidence is provided that MIP-1alpha directly contributed to CD4+ T cell recruitment and indirectly to PMN persistence in the infected cornea.

Evidence suggests that Pseudomonas aeruginosa stromal keratitis and corneal perforation (susceptibility) is a CD4(+) T cell-regulated inflammatory response following experimental P. aeruginosa infection. This study examined the role of Langerhans cells (LC) and the B7/CD28 costimulatory pathway in P. aeruginosa-infected cornea and the contribution of costimulatory signaling by this pathway to disease pathology. After bacterial challenge, the number of LC infiltrating the central cornea was compared in susceptible C57BL/6 (B6) vs resistant (cornea heals) BALB/c mice. LC were more numerous at 1 and 6 days postinfection (p.i.), but were similar at 4 days p.i., in susceptible vs resistant mice. Mature, B7 positive-stained LC in the cornea and pseudomonas Ag-associated LC in draining cervical lymph nodes also were increased significantly p.i. in susceptible mice. To test the relevance of these data, B6 mice were treated systemically and subconjunctivally with neutralizing B7 (B7-1/B7-2) mAbs. Treatment decreased corneal disease severity and reduced significantly the number of B7-positive cells as well as the recruitment and activation of CD4(+) T cells in the cornea. IFN-gamma mRNA levels also were decreased significantly in the cornea and in draining cervical lymph nodes of mAb-treated mice. When CD28(-/-) animals were tested, they exhibited a less severe disease response (no corneal perforation) than wild-type B6 mice and had a significantly lower delayed-type hypersensitivity response to heat-killed pseudomonas Ag. These results support a critical role for B7/CD28 costimulation in susceptibility to P. aeruginosa ocular infection.

Using a multiprobe RNase protection assay, we examined cytokine and chemokine mRNAs that were expressed after corneal infection with Pseudomonas aeruginosa in mice. Cytokines that were upregulated included interleukin-1alpha (IL-1alpha) and -1beta, IL-1 receptor antagonist, IL-6, IL-11, granulocyte colony-stimulating factor, granulocyte-macrophage colony-stimulating factor, macrophage colony-stimulating factor, stem cell factor, lymphotoxin beta, transforming growth factor beta1, and tumor necrosis factor alpha. Chemokine transcripts that were upregulated included Eotaxin; gamma-interferon-inducible protein 10; monocyte chemoattractant protein 1; macrophage inflammatory proteins 1alpha, 1beta, and 2; and RANTES. Peak expression of these cytokines and chemokines was observed between 1 and 3 days after infection. These responses returned to or approached baseline preinfection levels by 7 days after ocular challenge. Identification of the various cytokines and chemokines upregulated during corneal infection provides important information relevant to unraveling the pathogenesis induced by this bacterium and provides hope that specific molecules can be targeted for therapy.

Purified flagella from P. aeruginosa ATCC 19660 were used for active, passive, or topical immunization prior to corneal challenge with strain 19660. At 30 days post-infection, a significant number of mice actively or passively immunized with flagella and infected with the homologous bacterial strain were protected from ocular disease when compared to control animals. In topical immunization studies, premixing of 19660 flagella with the bacterial inoculum prior to ocular challenge with strain 19660, provided results similar to those of the active or passive immunization studies. A reduced lipopolysaccharide (LPS:1 E.U./mg) flagella preparation was also produced and used similarly. Again, significant protection was achieved in mice immunized by flagella regardless of the immunization route. An in vitro adherence assay also was performed to examine quantitatively the effect of exogenously applied flagella, or an antiflagella monoclonal antibody (MAb) on bacterial adhesion. Premixing of the bacterial inoculum with flagella or the MAb prior to applying it topically to corneas in organ culture all significantly inhibited bacterial binding. These results strongly suggest that significant ocular protection is achieved with either active or passive immunization, or premixing of the bacterial inoculum with flagella from strain 19660 prior to ocular challenge with the homologous bacterial strain. They also indicate that topical application of flagella or antiflagella MAb provide protection against ocular disease by decreasing bacterial adhesion to cornea.

The studies described here are aimed at determining the kinetics of antibody responses specific to Pseudomonas aeruginosa ATCC 19660 in sera, tears, and corneas of naturally resistant DBA/2 mice and susceptible C57BL/6 mice after intracorneal infection. Immunoglobulin (IgG) and IgM responses in sera were significantly greater in DBA/2 mice for the first 2 weeks postinfection. Little or no IgA was detected in the sera of mice from either strain. IgG was the predominant immunoglobulin class present in the corneas of the infected eyes from both mouse strains. However, differences in both the magnitude and the kinetics of the corneal IgG responses were noted between mouse strains. The kinetics of the corneal IgG responses were more similar to those of the serum IgG response than to those of the tear IgG response. Tear antibody responses in DBA/2 mice differed from those of C57BL/6 mice in two ways. First, there was a sharp increase in tear IgG levels 2 weeks after infection in DBA/2 mice that was not present in C57BL/6 mice. Second, IgA levels present in tears from the infected eyes of C57BL/6 mice dropped to nearly preinfection levels after the first week, whereas in DBA/2 mice, IgA levels remained elevated in the infected eyes after the first week. Determination of P. aeruginosa-specific antibody responses in the uninfected, contralateral control eyes revealed that IgA was detectable in the tears but not in the corneas of DBA/2 mice. Very little IgA was detected in the tears of the uninfected eyes of C57BL/6 mice. IgG was the only immunoglobulin class present in the uninfected corneas in both mouse strains tested. These results suggest that ocular IgA was made locally, whereas most ocular IgG may have originated from the serum, with some possible local synthesis. These immunological results indicate that DBA/2 and C57BL/6 mice respond differently to corneal challenge with P. aeruginosa.

Previous studies in our laboratory have indicated that naturally resistant, inbred DBA/2J mice mount a greater serum antibody response to Pseudomonas aeruginosa 19660 than susceptible C57BL/6J mice. However, the specificity of the antibody produced was not known. The present study examines the specificity and kinetics of the humoral response of these mouse strains to potential virulence factors produced by the organism during both a primary and a secondary corneal infection administered 4 weeks after the primary infection. Serum antibody levels specific for lipopolysaccharide (LPS), exotoxin A, phospholipase C (PLC), alkaline protease, elastase, and flagella were measured by enzyme-linked immunosorbent assay. Little or no antibody to either alkaline protease or elastase was detected during either primary or secondary infection. Immunoglobulin G (IgG) antibodies specific to exotoxin A, PLC, and flagella were detected 2 weeks after primary infection, and a rapid response to these antigens was measured 1 week after secondary infection. During primary infection, detectable LPS-specific antibody was only IgM, while IgG appeared only after secondary infection. The kinetics of the humoral response in susceptible C57BL/6J mice were similar to those in resistant DBA/2J mice, although the magnitude of the response varied according to the antigen tested. These results indicate that LPS, exotoxin A, PLC, and flagella are present or produced in amounts that are immunogenic during corneal infection by P. aeruginosa 19660 in the mouse strains tested.

The role of bacterial piliation and protease production in Pseudomonas aeruginosa adhesion to the injured corneal epithelial surface and subsequent infectivity was examined using several bacterial strains, including three that were hyperpiliated. To initiate this study, bacteria were examined by transmission EM to confirm their piliation characteristics. The PAK strain, like pseudomonas ATCC 19660, possessed about 1-4 polar pili. The mutant PAK/PR11 lacked pili while PAK/PR1, DB2, a mutant of PAO1, and PA1244, a wild-type clinical isolate, were hyperpiliated. Ocular infectivity of these bacterial strains and mutants was examined macroscopically and histopathologically in mice and these data compared to the well-characterized ocular disease response of a murine model of infection with pseudomonas ATCC 19660. The PAK strain was infective, but less virulent than strain 19660 by both macroscopic grading and histopathological analysis of infected eyes. Infectivity of the PR11 mutant was similar to the PAK parent strain, while PR1, DB2 and 1244, all hyperpiliated, were not infective. To explore the hypothesis that hyperpiliated bacteria bound less well to cornea and thus failed to induce corneal disease, in vitro quantitative studies of bacterial adhesion were done using an ocular organ culture model. The PR1 hyperpiliated mutant bound significantly less well to cornea than the PAK parent strain, PR11 mutant or pseudomonas 19660, while DB2 and 1244 binding did not differ significantly from 19660 or PAK. Examination of protease production, another factor which may influence adhesion, revealed that only 19660 and DB2 produced detectable protease. This study provides evidence that non-piliated, non-protease producing strains such as PAK/PR11 possess alternate virulence mechanisms to facilitate binding to and infectivity of corneal tissue.

Six- to eight-week-old BALB/cJ (and BALB/cPi) mice were found able to restore corneal clarity within 3 to 4 weeks after intracorneal infection with Pseudomonas aeruginosa strain 19660. However, enzyme-linked immunosorbent assay (ELISA) for serum immunoglobulins directed specifically against P. aeruginosa indicated that the mice were initially non- or hypo-responsive for IgG and IgA over the 4-week holding period. Low IgM titers could be detected 1 week after infection, but tended to decrease with time. When the mice were re-infected by using the contralateral control eye, then the serum IgG levels began to gradually increase as the time interval following the secondary infection increased from 1 to 3 weeks. Re-infected mice did not show a significantly increased rate of corneal clarity restoration with time, when compared to the corneas of mice receiving only a primary infection despite the presence of serum antibodies specific to P. aeruginosa. When congenic mice of the BALB/c background carrying the DBA/2N Idh/Pep-3 locus found on chromosome 1 were intracorneally infected, they tended to restore corneal clarity at approximately the same rate as the BALB/cJ mice. However, the congenic mice mounted a faster and substantially greater magnitude of serum IgM and IgG response during the primary infection than BALB/cJ mice receiving either a primary and/or secondary infection. IgG subclass studies with the congenic mice indicated that serum IgG1, and IgG2a to a lesser extent, were the primary immunoglobulins produced and no major shift in subclass was noted with time during the primary infection.(ABSTRACT TRUNCATED AT 250 WORDS)

The morphology, distribution and quantitation of dendritic (Langerhans) cells (LC) was determined by analysis of ADPase stained epithelial flat mounts from 6-8 week young adult (resistant) and 24 month old (susceptible) aged mice before and after experimental infection with P. aeruginosa topically applied to the scarified cornea. The contralateral eye (controls) was also scarified and phosphate buffered saline applied similarly. This study has examined the changes in ADPase positive cell populations of the conjunctival limbal epithelium and corneal epithelium of naturally resistant mice (Swiss-Webster and CD2F1) following corneal infection with Pseudomonas aeruginosa at two different ages, young adult (8 week old) and aged (24 month old). The young adult mice recover from their infection and restore corneal clarity while the aged mice have extensive ocular destruction and corneal scarring. Conjunctival limbal dendritic cell numbers in young adult mice were found to be significantly increased at day seven post infection and then returned to baseline levels. In contrast, conjunctival limbal dendritic cell numbers in aged mice were found to increase slowly and to peak at fourteen days after infection. Other differences between the two ages (young adult and aged) included an initial increase in dendritic cells five hours post infection in the young adult groups and an initial decrease at five hours in the aged groups of mice.

BALB/c-derived athymic, nude (nu/nu) mice exhibited a heightened natural resistance to experimental corneal infection with Pseudomonas aeruginosa when compared with their heterozygote (nu/+) littermates. Stereomicroscopic examination of the eyes of nu/nu mice 24 h after corneal trauma and topical bacterial application revealed slightly cloudy corneas (iris visible), whereas nu/+ littermate corneas were opaque (iris not visible). Nu/+ mice failed to resolve the infection, and endophthalmitis and shrinkage of the infected eye occurred in these mice within 2 weeks after experimental Pseudomonas infection, as in the parent BALB/c strain. However, nu/nu mice, similarly infected, resolved the infection within 24 h and never exhibited full corneal opacity or eye shrinkage. Histological examination of the corneas of nu/nu mice 24 h after experimental wounding and bacterial application demonstrated subepithelial capillaries and a few polymorphonuclear neutrophils (with numerous intracellular bacteria) in the central cornea. In contrast, the equivalent corneal areas of infected nu/+ littermates, examined similarly, showed a more striking neutrophilic response (but with few intracellular bacteria) to similar bacterial infection, as well as a lack of blood vessels within the central cornea. The central corneas of uninfected and saline control nu/nu mice also were observed. This area in nu/nu mice exhibited an infrequent polymorphonuclear neutrophil (with no intracellular bacteria) and capillaries similar in size and location to those described for experimentally infected nu/nu mouse corneas. Untreated and saline control nu/+ mice, on the other hand, lacked both vessels and polymorphonuclear neutrophils in the central cornea.

The murine genetic control of resistance to Pseudomonas aeruginosa eye infection previously has been demonstrated to be regulated by two complementing dominant genes, PsCR1 and PsCR2. The PsCR1 locus apparently is not associated with the H-2 complex, whereas the PsCR2 locus could not definitively be associated with H-2. In this study we attempted to demonstrate a possible H-2 linkage of the PsCR2 locus. A panel of inbred congenic strains varying with either the H-2 haplotype or genetic background from inbred partners of C57BL/10, C3H, A, and BALB/c strains were characterized for their P. aeruginosa infectivity phenotypes. These studies indicated that the PsCR2 locus is not associated with the H-2 locus. Furthermore, variations of the H-2 haplotype did not change the resistance patterns observed in these strains. However, BALB.B and BALB.K congenic lines were resistant to P. aeruginosa eye infection, whereas BALB/cJ mice were susceptible. Examination of hybrids (BALB.K X BALB/cJ)F1 and (BALB.B X BALB/cJ)F1 demonstrated that an autosomal dominant gene(s), PsCR, confers resistance. Segregation analysis for the H-2 haplotype and the PsCR gene in offspring of backcross matings with the BALB/cJ parental strain suggested that this PsCR gene is not linked to the H-2 complex and has an inheritance pattern of a single locus or several tightly linked loci.

Natural host resistance to Pseudomonas aeruginosa corneal infection is regulated by two complementing dominant genes PsCR1 and PsCR2 (RS Berk, MA Leon, LD Hazlett. Infect Immun 26:1221-1223, 1979). In this study we have demonstrated a third dominant gene, which determines susceptibility to P. aeruginosa-induced eye damage. This gene was designated as PsCS. The F1 progeny from matings between the resistant DBA/2J strain and the susceptible strain C57BL/6J and C3H/HeJ displayed the susceptibility phenotype. Backcross and F2 studies using the C3H/HeJ and DBA/2J strains suggested the presence of two linked PsCS loci.

Scanning and transmission electron microscopy were used to characterize the normal pup and adult mouse ocular surface. Various fixatives were examined and categorized into those which stabilize and preserve ocular mucus associated with surface corneal epithelial cells and those which do not, but allow good visualization of surface detail for scanning electron microscopy. Desquamation patterns of corneal epithelial surface cells in the mouse pup and adult were examined, compared with each other and with desquamation patterns originally reported for several other animal species. Once the normal cytoarchitecture of the corneal surface and its associated ocular mucus was established for the mouse pup and the adult animal, the murine ocular response to P. aeruginosa infection was studied. Those studies revealed that in the pup, after inoculation of bacteria beneath the eyelid, with no corneal scarification, organisms adhered preferentially to young surface cells and quickly penetrated the corneal and conjunctival epithelium. In contrast, scarification of the corneal surface had to precede topical bacterial inoculation for infection to occur in the adult cornea. In this model, bacteria adhered initially to the wound site and to aged cells but did not preferentially adhere to young surface cells. An extracellular virulence factor, exotoxin A, produced by the bacteria, was also examined to determine its role in P. aeruginosa pathogenesis. Studies using toxin A, have shown that it was capable of inducing epithelial and endothelial cell death and corneal necrosis in the adult scarified cornea. In the pup, where no scarification preceded toxin A challenge, little corneal epithelial damage was produced when compared with the infection model. Nonetheless, as with bacterially challenged pups, experimental toxin A treated animals died within 24 hr following toxin administration.

Male rats that consumed liquid alcohol diets containing 35%, 17.5 or 0% ethanol-derived calories for a minimum of 3 weeks were bred to females which were fed similar diets during pregnancy. At approximately 50 days of age, offspring were challenged with 10 X 10(7) Pseudomonas aeruginosa onto the scarified cornea. The ocular response, evaluated macroscopically, for 3 weeks, revealed a significant dose-related effect of both maternal and paternal alcohol exposure. The higher the parental alcohol consumption the earlier and the more frequently the cornea of progeny perforated. There was no effect of sex of offspring or interaction between maternal and paternal factors. Histopathology confirmed the above data in that progeny of parents receiving 0% or lower-dose alcohol treatment had less severe corneal pathology than progeny of parents with higher alcohol doses.

The ocular damage in mouse pups produced by purified Pseudomonas aeruginosa exotoxin A was examined microscopically following toxin A injection beneath the fused eyelid. In contrast to infection, in which lysis of ocular surface epithelium occurs within minutes, toxin A does not induce any lytic changes until 5 h after injection. Additionally, no phagocytic cellular response to toxin A was observed, whereas in infection, this response is seen within 1 h. We conclude that toxin A is not the bacterial extracellular factor responsible for mediating the rapid early destruction of the ocular epithelium allowing bacterial penetration and dissemination.

The ocular histopathological response of C3-depleted and normal DBA/2J mice was compared after corneal scarification and experimental infection with topically applied Pseudomonas aeruginosa. Normal, non-C3-depleted mice were capable of mounting a vigorous polymorphonuclear leukocyte response at 24 h after bacterial challenge and eventually restored corneal clarity. In contrast, C3-depleted animals responded at 24 h to the ocular bacterial challenge with a decreased number of polymorphonuclear leukocytes migrating into the cornea. Additionally, the eyes of C3-depleted mice exhibited persistence of bacteria, cataract of the ocular lens, and loss of vision.

This study provides evidence for an N-acetylmannosamine (manNac) receptor mediating in vivo bacterial adherence of P. aeruginosa to the scarified corneal epithelial surface. Bacteria, whether exposed to the inhibiting sugar or not, did not adhere immediately after inoculation, but required time in contact with the scarified corneal surface to adhere and adherence increased with time. Organisms were observed primarily adherent to the surface of epithelial cells lining the wound and were less frequently seen on areas of denuded stroma. Saturation of binding sites on the bacterial surface by manNac inhibited attachment of the organisms to newly exposed surface membrane receptors. In vivo protection studies showed excellent correlation with quantitative analysis of scanning electron micrographs, in that the number of adherent organisms at the corneal surface at 60 min following scarification and bacterial inoculation was decreased significantly by prior treatment of the bacteria with manNac when compared with PBS or the other monosaccharides. These data were confirmed by quantitative bacterial culture of excised corneas which had been similarly infected in vivo with PBS or manNac treated organisms.

The ocular damage in young adult mice produced by purified Pseudomonas aeruginosa exotoxin A was microscopically characterized at 1 and 5 h and at 1, 3, 5, 7, 10, 14, and 21 days after toxin A challenge, using light, transmission, and scanning electron microscopic techniques. Similarly to previously described infection with viable organisms, toxin A killed both epithelial and endothelial cells and induced stromal cell swelling within 5 to 24 h after application onto the nonpenetrating wounded corneal surface. Other toxin-induced damage similar to the damage produced by infection with the viable bacteria was production of electron-dense particles within the corneal stroma, dispersal of undamaged collagen fibrils, and apparent loss of stromal proteoglycan ground substance. Toxin A damage differed from infection with the viable bacteria in essentially two ways. First, more purulent exudate and more polymorphonuclear neutrophilic leukocyte (PMN) infiltration of the corneal stroma were produced by infection with the viable organisms than by the toxin. Additionally, PMN did not appear within the toxin-treated corneas until 3 days after treatment, whereas in corneas infected with the viable organisms, PMN were numerous by 18 h. Secondly, toxin A produced cataract of the ocular lens, whereas infection with the viable organisms did not.

Inbred mouse strains differ in susceptibility to intracorneal challenge with Pseudomonas aeruginosa. Genetic studies indicate that resistance to corneal infection is dominant over susceptibility and is controlled by autosomal genes, at least one of which is located outside of the H-2 locus. On the basis of genetic complementation studies, the susceptible strains BALB/c and C57BL/6 each bear one resistance gene, since the F1 hybrid (BALB/c X C57BL/6) was uniformly resistant to infection.

We studied the responses of mice to ocular challenge with purified exotoxin A from Pseudomonas aeruginosa in 5-, 10-, 16-, 21-, and 30-day-old animals. In the absence of trauma, injection of 3 to 6 microgram of exotoxin per mouse beneath the fused eyelids of 5-day-old Swiss-Webster mice resulted in death of all animals within 24 h. Administration of 1.5, 0.75, and 0.375 microgram of exotoxin per mouse resulted in 24-h mortality rates of 50, 22, and 20%, respectively. Additional deaths were recorded throughout the next 4 days. Similar lethality results were obtained with 10-day-old animals that received equivalent amounts of exotoxin beneath the fused eyelid and in these experiments, the 72-h 50% lethal dose was 0.49 microgram of exotoxin per mouse. Mice that were 16 and 21 days old, whose eyelids were open, each received from 0.375 to 15 microgram of exotoxin topically applied to the surface of a wounded cornea. Cataracts were observed within 1 week in both groups, and none of the animals that received the higher concentrations of toxin died. Young adult (30-day-old) animals also received from 1.8 to 15 microgram of exotoxin topically on the surfaces of wounded corneas. Corneal swelling and slight opacity were observed at 24 h and within 1 month; 80% of these mice had cataracts of the ocular lens.

The purpose of this study was to compare and characterize histopathologically the virulence of two bacterial strains in a naturally susceptible animal host. One of the strains Pseudomonas aeruginosa ATCC 19660, routinely used in this laboratory, produces endopthalmitis and phthisis bulbi in C57BL/6 mice within 3-4 weeks. The other bacterial strain, a clinical isolate from a 70-year-old patient with Pseudomonas corneal infection was found to be less virulent in the patient and thus was examined for virulence in the susceptible mouse model. The strain also proved to be less virulent in the normally susceptible mouse when compared with ATCC 19660. In general, when comparing the histopathological response of the mice to the two bacterial strains, it was found that in contrast to ATCC 19660, fewer polymorphonuclear neutrophils migrated into the cornea at 24-72 h, endothelial and epithelial necrosis was delayed, few free bacteria were observed in the cornea and animals recovered corneal clarity by 21-25 days following infection.