Study on antimicrobial potential of neem oil nanoemulsion against Pseudomonas aeruginosa infection in Labeo rohita Prabhakar Mishra Suresh Kumar R. S. Jayakumar Jerobin John Thomas Amitava Mukherjee Natarajan Chandrasekaran * Centre for Nanobiotechnology, VIT University, Vellore, Tamil Nadu, India Abstract Presence of several biochemical constituents in neem makes it an efficient antimicrobial agent for pathogenic diseases. The current investigation was aimed to assess the therapeutic potential of neem nanoemulsion as a control measure for Pseudomonas aeruginosa infection in freshwater fish Labeo rohita. The median lethal concentration (LC 50 ) for the neem oil and neem nanoemulsion was 73.9 and 160.3 mg/L, respectively. The biomarker enzymes of treated fish tissues showed a significant difference in the level of glutathione reductase, catalase, and lipid peroxidation in neem oil-treated samples than in neem nanoemulsion-treated samples at P < 0.05. The results were corroborative with histopathology and ultrastructural analysis. The bacterial infection of P. aeruginosa treated using neem nanoemulsion was more effective in both in vitro and in vivo methods. Present findings suggest that neem-based nanoemulsion has negligible toxicity to Rohu fishes. This makes neem-based nanoemulsion as an efficient therapeutic agent against P. aeruginosa infection, leading to its possible usage in the aquaculture industry. C  2014 International Union of Biochemistry and Molecular Biology, Inc. Volume 61, Number 5, Pages 611–619, 2014 Keywords: biosafety, histopathology, neem nanoemulsion, Labeo rohita, Pseudomonas aeruginosa 1. Introduction Aquaculture industries have emerged as one of the most promising and fast growing industries providing high-quality animal proteins with nutritional level and also tend to generate income and employment across the globe. Aquaculture sce- nario in India describes the production of three major carps (Labeo rohita, Catla catla, and Cirrhinus mrigala). The global production of carp species such as L. rohita was around 1.2 million tons in 2005, out of which, 1 million ton is contributed Abbreviations: CFU, colony forming unit; DMSO, dimethyl sulfoxide; EPA, Environmental Protection Agency; GSH, reduced glutathione; GSSH, oxidized glutathione; MTCC, Microbial Type Culture Collection; PBS, phosphate-buffered saline; ROS, reactive oxygen species; TEM, transmission electron microscopy; TBARS, thiobarbituric acid reactive substances. ∗ Address for correspondence: Natarajan Chandrasekaran, PhD, Senior Professor & Director, Centre for Nanobiotechnology, VIT University, Vellore 632014, India. Tel.: +91 416 2202624; Fax: +91 416 2243092; e-mail: nchandrasekaran@vit.ac.in; nchandra40@hotmail.com. Received 20 November 2013; accepted 1 February 2014 DOI: 10.1002/bab.1213 Published online 19 September 2014 in Wiley Online Library (wileyonlinelibrary.com) by India [1]. The pathogenic diseases are one of the frequent problems occurring in modern aquaculture system. At present intensive aquaculture system, ponds are being fertilized using inorganic chemicals; they are supplemented with artifcial feed and stocking fsh at high densities. All these innovative practices in the aquaculture lead to increment in the fsh production from particular ecosystem and at the same time causing stress conditions to fshes. Fishes are liable to various infections. High content of organic load-contaminated feed and unhygienic conditions are some of the important factors leading to an outbreak of bacterial diseases in aquatic animals [2]. Pseudomonas is one of the bacterial species that are frequently associated with fsh diseases [3]. Pseudomonas infection in fsh leads to the development of so-called red skin disease, which occur throughout the year particularly when fsh is injured either because of inappropriate handling or during transportation. The prevailing lacunae in means of disease control often lead to high mortality, resulting in heavy losses. Neem oil plays an infuential role as an effective natural pesticide having a strong antifeedant and growth regulating properties in it [4]. There are many reports suggesting the side effect of neem pesticides in terrestrial ecosystem than aquatic ecosystem [5]. Advancement of nanoscience and nanoscale techniques in recent times holds the promise of target de- livery, better stability, nutrient utilization, pest control, and 611