This work was supported by a grant from Medical Biotechnology Research Center, School of Paramedicine, Guilan University of Medical Sciences, Rasht, Iran. Conflicts of Interest The authors declare no conflict of interest regarding the publication of this article.. Therefore, the presentation of new adjunctive prophylactic/therapeutic modalities seems to be still necessary (3). Infection of the cornea by bacteria occurs when the epithelial barrier function becomes injured or compromised such as mild chronic damage caused by contact lens wear. In such situation, as an opportunistic pathogen could access to the epithelial layer and cause infection (4). Similar to other mucosal surfaces including urinary tract, airway, and intestinal, Toll-like receptor 5 (TLR5) acts as the major pathogen recognizing receptor (PRR) at the surface of the cornea (5). As the first line of defense, ocular epithelium recognizes and responds to the motile pathogen via TLR5 which activates mucosal innate defense by producing proinflammatory cytokines leading to employ polymorphonuclear leukocytes (PMNs) to the infection site. PMNs by the secretion of antimicrobial agents, directly eradicate the invading bacteria (6). The vital role of PMNs in the elimination of has been documented (7). TLR5-knockout mice are more susceptible to ocular infection (8), indicating the role of TLR5 in the forming of protective mucosal surfaces against the infection by flagellated FSCN1 bacteria. Single polar flagellum-mediated motility plays an essential role in the pathogenesis of gene) is classified into two distinct types: a-type and b-type flagellins. This classification is based on molecular weight and reactions with specific antibodies. The type a-flagellins (FLA) are a heterologous group with molecular weight of 45-52 kDa, whereas type b-flagellin (FLB) is a homologous group having a molecular weight of 53 kDa (12). Each strain only possess one type of flagellin, so that, its gene does not undergo antigenic variations. Flagellin has been utilized as a vaccine candidate MK-8745 against in different animal models: FLB protected mice in lung infection (13, 14), urinary tract infection (UTI) (15) and keratitis (16, 17). Also, immunization with FLA afforded protection in lung infections (18). Our previous work showed that active and passive immunization with FLA provided protection against in the burn mouse model (19). Divalent flagellin (FLA and FLB) preparation successfully confined burn wound infections (20), UTI (21) and cystic fibrosis (CF) (22). To our knowledge, the therapeutic effects of antibodies raised against divalent flagellin for keratitis has not been reported yet. In the present study, we assessed whether topical administration of antibodies to divalent flagellin (FLA and FLB) can have therapeutic efficacy in the mouse model of keratitis. Materials and Methods PAO1 (kindly provided by Dr. Abdi from Department of Microbiology, Al-Zahra University, Tehran, Iran) and a type a-flagellated strain of PAK (available in our laboratory) were used. The strains were controlled for purity and identified before using in this study. They were cultured in Luria Bertani (LB) broth (HiMedia, India) and maintained in 20% glycerol and kept at -20 C. gene was obtained from strain HMSC05H02 (Sequence ID: “type”:”entrez-protein”,”attrs”:”text”:”KJJ22056.1″,”term_id”:”768692735″,”term_text”:”KJJ22056.1″KJJ22056.1). The NcoI and XhoI sites were located at the 5 and 3 ends of the gene, respectively. The coding gene was designed into the expression vector pET28a to produce the recombinant pET28a/BL21 (DE3) and finally purified by Nickel-affinity chromatography. were grown at 37 C in Peptone Tryptic Soy Broth (PTSB, Difco Laboratories, Detroit, MI) for 18 hr and the OD650 nm adjusted to inocula of about 3 107 CFUs/eye. Female BALB/c mice anesthetized with ketamine and xylazine (Alfasan, Woerden-Holand) were placed beneath a stereoscopic microscope and then three 1-mm incisions were made on the eyes using a 26-gauge needle, and finally the bacterial strains (in a 5 l volume) were inoculated in the injured site. The disease progression was daily monitored with a dissection microscope equipped with a digital camera. strains PAK and/or PAO1, and the viable bacteria were calculated using plate count technique. Individual corneas were homogenized in Tryptic soy broth including 0.5% Triton X-100, and diluted and cultured onto Cetrimide (strains PAK and/or PAO1 for 48 hr. To measure the extracellular LDH, the MK-8745 infected corneas were collected and washed with PBS including PMSF (as protease inhibitor). By using a cytotoxicity detection kit plus (Roche Diagnostics, Mannheim, Germany), the extracellular LDH release was detected. opsonophagocytosis test was done according to the protocol of Faezi (23). In brief, 100 l of bacterial inoculum [~ 2 109 CFUs/ml of PAK or PAO1 in 1% bovine serum albumin (BSA)] was exposed to the same volume of heat-inactivated four two fold dilutions (1:4 to 1 1:64) of specific anti-FLA and anti-FLB IgG at 22 C for 60 min. Then, it was washed two times MK-8745 with BSA [1% (w/v)] to remove excessive antibodies. One hundred.