Short communication In vivo and in vitro antimicrobial activity of Azadirachta indica (Lin) against Citrobacter freundii isolated from naturally infected Tilapia (Oreochromis mossambicus) S. Thanigaivel, S. Vijayakumar, S. Gopinath, Amitava Mukherjee, Natarajan Chandrasekaran, John Thomas ⁎ abstract article info Article history: Received 28 November 2014 Received in revised form 3 December 2014 Accepted 5 December 2014 Available online 12 December 2014 Keywords: Oreochromis mossambicus Natural infection Citrobactor freundii Medicinal plants Plant extracts 16s rRNA sequencing Aquaculture health management demands a constant growth in the production by stabilizing economic loss and implementing various natural plant-based bio products for treating pathogenic bacteria. Pathogenic effects of the Citrobacter freundii isolated from fingerlings of Tilapia collected from a fish farm showed symptoms like tail necrosis, septicemia, hemorrhage, and reddening of the body. Antibacterial activity of C. freundii was tested against various medicinal plants. Of the three plants screened, Azadirachta indica (Neem) showed good antibac- terial activity against C. freundii. The present study revealed that A. indica plant extract at a concentration of 150 mg/l in vivo can be used as an alternative to antibiotics for treating this bacterial infection. The use of medic- inal plant extract-mediated treatment was found to be very effective in controlling this bacterial infection. © 2014 Elsevier B.V. All rights reserved. 1. Introduction Health management in the aquaculture industry is a key factor for the successful production of aquatic animals and their products, which can be achieved by efficient disease management with the help of vaccines and effective formulation of extracts and emulsion prepara- tions (Austin and Austin 1999; Thanigaivel et al. 2014; Thomas et al. 2014). Plant-based extracts are now being used to treat and control diseases in aquaculture instead of commercial antibiotics which cause resistance against the particular pathogenic bacteria (Mirand and Zemelman, 2002; Seyfried et al. 2010). Moreover, the regular consump- tion of antibiotic treated fishes by humans can lead to complications. In addition, it also makes fishes more immune compromised against the particular opportunistic pathogenic bacteria (Cabello 2006; Romero Ormazábal et al., 2012). The evolving method of using plant-based extracts for treating various fish diseases has been considered to be best alternative for controlling the spread of infection by increasing the fish and shrimp immunity and the innate behavior of fish (Reverter et al 2014., Citarasu, 2010., Lee et al. 2009; Makkar et al. 2007; Mohapatra et al. 2013; Panigrahi and Azad 2007). There are many recent reports that have been published in the field of medicinal plants based their antibac- terial and antimicrobial activity (Khan et al 2010). In recent years, di- verse formulations of plant extracts with many chemically identified compounds and biologically active molecules have been recommended for commercial use (Acharyya et al 2009, Ogbulie et al 2007, Toranzo et al 2005.) The in vivo treatment of bacterial diseases in aquaculture using plant extracts is based on the following: (1) selecting the appropriate optimized extraction procedure, (2) choosing the desired dose for administration (3) Mechanism of action. This approach will be helpful in treating bacterial diseases in aquaculture (Kumar et al. 2013). The use of plant extracts by the traditional extraction method is employed for the eco friendly approach and cost-effectiveness method and does not cause side effects. The aim of the present work was to study the antibacterial effect of the aqueous extract of four plants (Azadirachta indica, Cynodon dactylon, Ocimum tenuiflorum, and Phyllanthus niruri) on Citrobacter freundii isolated from fingerlings of Oreochromis mossambicus. 2. Materials and methods 2.1. Collection and maintenance of naturally infected animals Diseased fingerlings of tilapia (O. mossambicus), which were exam- ined based on the clinical symptoms such as septicemia, hemorrhagic lesions, and ulcer were collected from a fish farm located near Walajapet, Vellore district, Tamil Nadu, for experimental purpose between August 2013 and December 2013. Their average body weight was 10–15 g. Live fishes were transported and maintained under labo- ratory condition with proper aeration in a 1000 l tank. They were then used for the screening of different bacterial pathogens. Aquaculture 437 (2015) 252–255 ⁎ Corresponding author at: Centre for Nanobiotechnology, VIT University, Vellore, Tamil Nadu, India. Tel.: +91 416 2202876; fax: +91 416 2243092. E-mail addresses: john.thomas@vit.ac.in, th_john28@yahoo.co.in (J. Thomas). http://dx.doi.org/10.1016/j.aquaculture.2014.12.008 0044-8486/© 2014 Elsevier B.V. All rights reserved. Contents lists available at ScienceDirect Aquaculture journal homepage: www.elsevier.com/locate/aqua-online