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Year : 2018  |  Volume : 13  |  Issue : 4  |  Page : 383-391

Ex vivo caprine model to study virulence factors in keratitis

Department of Microbiology and Biotechnology Centre, Faculty of Science, The Maharaja Sayajirao University of Baroda, Vadodara, Gujarat, India

Correspondence Address:
Devarshi Urvish Gajjar
Department of Microbiology and Biotechnology Centre, The Maharaja Sayajirao University of Baroda, Vadodara 390 002, Gujarat
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Source of Support: None, Conflict of Interest: None

DOI: 10.4103/jovr.jovr_131_17

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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.

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