Ex vivo caprine model to study virulence factors in keratitis

Abstract

Purpose:To develop an infectious keratitis model using caprine (goat) corneas and to investigate the expression of virulence factors during infection.Methods:Goat eyes were surface-sterilized and dissected, and the corneas were placed on an agarose-gelatin solid support (0.5% in phosphate-buffered saline) in a 12-well culture plate containing 10% fetal bovine serum-supplemented culture medium for 3 weeks. Cell viability tests (trypan blue and MTT) were performed on the cultured corneas. Corneas were infected with Pseudomonas aeruginosa and Fusarium solani separately. Infection progression was observed via histological analysis and hematoxylin and eosin (H-E) staining. For Pseudomonas-infected corneas, expression of eight virulence genes (exoS, exoT, exoY, alpR, prpL, lasA, lasB, and algD) was determined via quantitative real-time PCR (qRT-PCR) at 48-h and 72-h time-points. For Fusarium-infected corneas, expression of five proteases (C7Z0E6, C7ZFW9, C7Z7U2, C7ZNV5, and C7YY94) was quantified via qRT-PCR at 2, 4, and 8 days after infection. Protease from infected corneas was detected via gelatin zymography.Results:Goat corneas with a viable epithelium could be maintained for 15 days. Pseudomonas infection progressed rapidly, and complete corneal degradation was observed on day 4 after infection. Fusarium infection progressed more slowly. Histological analysis and H-E staining of Fusarium-infected cornea revealed mycelia penetrating all layers of the cornea. qRT-PCR revealed expression of all eight virulence factors, and statistically significant difference in expression of prpL and alpR in Pseudomonas-infected corneas. Expression of C7ZNV5 was highest in Fusarium-infected corneas.Conclusion:Goat corneas can be used to evaluate the expression of virulence factors involved in Pseudomonas and Fusarium infection.

Corneal Infection Models: Tools to Investigate the Role of Biofilms in Bacterial Keratitis

“…A lack of standardised methods and paucity of information on animal models means comparing ex vivo studies is difficult, and there is dispute regarding the suitability of different animal models. Ex vivo models used to investigate bacterial keratitis include mice [ 137 , 138 , 139 , 140 , 141 ], rabbits [ 140 , 141 , 142 , 143 , 144 , 145 , 146 ], goats [ 147 ], cows [ 148 ] and pigs [ 149 , 150 , 151 ]. It is currently unknown if interspecies differences in the thickness of the corneal epithelium [ 108 , 152 ] and stroma [ 153 , 154 , 155 ] play a major role in development and progression of infection in the ex vivo cornea.…”

“…Various techniques have been used to induce bacterial infection in ex vivo corneas, including prolonged exposure to bacteria [ 166 , 167 ], use of infected contact lenses [ 149 ], superficial injury (e.g., tissue paper blotting) [ 168 ], corneal scarification [ 146 , 147 , 151 ] and intrastromal injection [ 143 ]. Differences in infection method, inoculum size, culturing techniques, incubation times and bacterial strains mean that comparing ex vivo studies is challenging.…”

“…In agreement with Pinnock et al, we recently demonstrated that there was no significant difference in viable cell count between ex vivo porcine and rabbit cornea models after 24 h infection, nor when two different strains of P. aeruginosa were used [ 151 ]. Furthermore, while some studies have reported that infection in ex vivo corneas is easy to establish and that progress is visible within less than 24 h [ 143 , 151 , 167 ], Madhu et al found that incubation time could be extended by a few days if a smaller inoculum was used [ 147 ]. Despite issues with standardisation, ex vivo models have been used to study various aspects of bacterial keratitis.…”

Identification of Proteases: Carboxypeptidase and Aminopeptidase as Putative Virulence Factors of Fusarium solani Species Complex

“…Ex vivo corneal infection model was previously developed in the lab (Madhu et al, 2018). It was performed with few modifications.…”

“…Media was changed after every 6 hours and was estimated for protease activity using azocasein assay. Histology of infected corneas at 8 th day was carried by method of Madhu et al, 2018. Briefly, the infected corneas were fixed with 2% buffered paraformaldehyde solution.…”