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ISBN 81-90 0947-3-7

RECENT ADVANCES IN LIFE SCIENCES Proceedings of XV AZRA International Conference, Ethiraj College for Women, Chennai, 11-13 February, 2016

Inaugural Session: From left Dr. M. Sujatha, Dr. M. Balasundaram, Dr. B.V. David, Thiru M.V. Muralidharan, Sri T. Rabikumar, Dr. Anand Prakash, Dr. A. Nirmala, Dr. R.W. Alexander Jesudasan, Dr. K. Revathi & Dr. PremaSampathkumar Editors

Anand Prakash, Jagadiswari Rao, K. Revathi Published by

Applied Zoologists Research Association (AZRA), Bhubaneswar, Odisha, India&

Ethiraj College for Women, Chennai, T.N.

ISBN 81-90 0947-3-7

RECENT ADVANCES IN LIFE SCIENCES Proceedings of XV AZRA International Conference,

Ethiraj College for Women, Chennai, 11-13 February, 2016 Editors

Anand Prakash Jagadiswari Rao K. Revathi Published by

Applied Zoologists Research Association (AZRA), Bhubaneswar, Odisha, India &

Ethiraj College for Women, Chennai, T.N.

2016 ISBN 81-90 0947-3-7 © AZRA-2016 RALS Book

Recent Advances in Life Sciences This is the Proceedings of XV AZRA International Conference on “ Recent Advances in Life Sciences” held on 11-13 February, 2016 at Ehtiraj College for Women, Chennai, Tamil Nadu, published by Applied Zoologists Research Association (AZRA), Bhubaneswar, India, Pages 250, HB, Price Rs.500/- in India and US$ 50/- for other countries excluding postage and handling charges Rs. 50/- and US$ 5. This book contains 43 selected research papers contributed and presented by the applied zoologists/biologists during the above mentioned conference & also the proceedings & recommendations of the conference. However, the editors and the publisher cannot assume responsibility for the validity of all materials or for the consequences of their use. Neither this book nor any part may be reproduced or transmitted in any form or by any means, electronic or mechanical, including photocopying, microfilming, and recording or any other information storage or retrieval system, without prior permission from the publisher. The consent of AZRA does not extend for copying for general distribution, for promotion, for creating new works, or for resale. Specific permission may be obtained from AZRA for such copying. Direct all inquiries to: Dr. Anand Prakash, General Secretary, Applied Zoologists Research Association (AZRA), K-9B/ 285 Bhagabanpur, Patrapada, Bhubaneswar -751 019, Odisha, India. Copy Right: Applied Zoologists Research Association, Bhubaneswar-753006, India

CONTENTS Foreword 1 Anand Prakash, Conference proceedings & recommendations 3 Col. (Dr.) C. P. Churamani, Prospectives of biothreats and their mitigation strategies 9 D. Loeto, E. Monthusi, B. Letsholo and K. Wale, Incidence and antimicrobial 17 susceptibility testing of Listeria monocytogenes in four street-food vending sites in Gabarone, Botswana A. Subashini and K. Revathi, Effect of immunostimulant on the physiological changes in 23 the kuruma shrimp, Marsupenaeus japonicus affected by white spot syndrome virus (WSSV) R.Sashindran, M.Balasundaram, Jegathambigai, and P. Kumar, Neuroprotective effect of 31 quercetin and coenzyme Q10 in ethanol induced neurotoxicity in mice Anil Kumar Dubey and Sudhir Singh, New record of the genus and species, 37 Agrostaleyrodes arcanusKo (Hemiptera: Aleyrodidae) from India, now colonizing on sugarcane R. K. Patel and L. V. Ghetiya, General inventory of odonates in south Gujarat, India 41 Sharmila Kumari. S. and Mohanasundari, Isolation and identification of antibiotic 49 producing soil bacteria and its antagonistic activity A. Lakshmi Devi, C. Vetriselvi and M. Sujatha,Phthalate screening in plastic users by 51 FTIR Spectroscopy P. Suresh, G. Sasireka and K.A.M. Karthikeyan,Genetic variability of populations of 55 Eariasvittella (Fab.) (Noctuidae: Lepidoptera) as revealed by Random Amplified Polymorphic DNA Amritha. N., K. Revathiand M. Babu,Effect of green algae (Chlorella vulgaris ) on the 59 production performance of Japanese quails (Coturnixcoturnix japonica) Revathi. K, Anjalai. K, Prabakaran. R., Babu. MandKirubakaran.R., Effect of green 63 microalgae (Chlorella vulgaris) on carcass characterstics of Japanese quail (Coturnixcoturnix japonica) P. Aswathi, Insecticide free home vegetable garden with the aid of bioagent, weaver ants 67 D. Anandhi and K. Revathi, 4, 8-dihydroxybenzo (de) chromen-2(4H)-one : Apurified 71 compound form Caesalpinia coriaria - Induction of Apoptosis and Cell Cycle Arrest in Human Carcinoma Cells (MCF - 7, SiHa and A-549 cell lines) P. B. Meshram, Eco-friendly approaches for the management of bark eating caterpillar, 79 Indarbela quadrinotata in Emblica officinalisplantations E. Logeswari, K.Revathi, G. Ananthan, and N. Arunagirinathan,Conventional taxonomic 85 identification of Ascidians from south coast of India GayatreeSahoo and B. K. Sahoo, Comparative efficacy of safer insecticides and plant 91 products against pod borer species of pigeonpea K. Revathi and A. Josephine, Comparative study of leaf, bark and heart-wood extracts of 97 Caesalpinia sappan for antiinflmatory activity L. K. Rath and SeemaTripathy, Effect of silicon on yellow stem borer of rice 105 M. Sasikala, N. Chitra and N. JothiNarendiran, Effect of an azo dye, methyl red on 109 biochemical constituents in muscle tissue of fresh water fish, Oreochromis mossambicus N. Punitha, R.Saravanan and N. Settu, Effect of semi ripe Caricapapaya fruit pup and 115 seed extracts on lipid constituents in reproductive structures of male albino rats A.D. Patel and L.V. Ghetiya, Population Fluctuation of Tetranychus urticae Koch in 123 Relation to Weather Parameters and Predatory Thrips on Marigold, Tagetesspp Priyankadevi, K. Revathi and C. Raghunathan,In Vitro Cell Culture analysis of 129 Plakobranchus ocellatus (Van Hasselt, 1824) from Andaman Islands, India Contd… Contents

Contents. Contd… R. Sangeetha, Anita R., J. Singh, and J. M. V. Kalaiarasi1, Biochemical changesnand antibiotic sensitivity test on Vibriosis-induced Penaeusmonodon R. Saravanan , S.Venkatesan and K.Revathi,Biochemical studies on the impact of tannery effluent and its phytoremediation effect in the fingerlings of fresh water fish, Channa striatus S. Mumtaj, G.B. Brinda Devi, P. Rajalakshmi and S. Sheela, Effective vaccine development against Columnaris disease from Flavobacterium columnare S. R. Ramya and K. R. T. Asha,Optimization and growth kinetics of Exopolysaccharide produced by moderately haemophilic bacteria, Virgibacillus marismortui Priya, J., K. Revathi, M. Babu and P. Shamsudeen, Effect of fermentation on antioxidant capacity of Panchagavya for its utilization in poultry feed S. Gowrilakshmi, M.S. Nalina Sundari and N. Deepa, Laboratory Assesment of Larvicidal Activity Using Culture Filtrate of Four Entomopathogenic FungI against rice moth, Corcyra cephalonica S. Sheela, G.B. Brindha Devi, P. Rajalakshmi and S. Mumtaj, Phytochemical and spectroscopic evaluation of medicinal plant, Reissantia indica C. Vetriselvi and K. Revathi, Effect of triazole fungicide tebuconazole on lipid metabolism in Labeo rohita – a fresh water fish S. Priya and Shyamala Kanakarajan, Effect of salt stress on growth and development of Lycopersicon esculentum mill K. Samyappan, T. Vijayakanth and D. Prabakaran, Impact of bilateral eyestalk ablation on a few biochemical constituents in freshwater male crab, Spiralothelphysa hydrodroma V. Manimozhi, Effect of fresh leaf extracts of Ocimum sanctum linn. on the somatic chromosomes of Allium cepa linn. Chauhan, Rinki Kumari and Abhishek Shukla, Bionomics of two spotted spider mite, Tetranychus urticae Koch. - a serious pest of french bean Nithya Sathiandran and Sabu, K. Thomas, Dung beetles (coleopteran: scarabaeinae) attracted to omnivore during in the forests in the moist south western ghats Sai Prathiba Avarsala, Malathi Veeramani, Revathi Kasthuri , Invitro and invivo antidiabetic activity of Linum usitassimum Mahadev Rama Kokane, Satyanarayan Sethi, R. Revathy and Anuradha, Population Studies of Ragged Sea Hare, Bursatella leachii in Pulicat Lake, Tamil Nadu S. Kavitha and S. S. Gnanamanickam, Effect of rhizosphere bacterial antagonists in combination with disease resistance genes in suppression of sheath blight and blast diseases of rice B. Thendral Hepsibha, C. Saravana Babu, V. Premalakshmi and T. Sekar, Effect of methanol leaf extract of Azima tetracantha Lam. against adjuvant arthritis, With special reference to lipid parameters M. Sujatha, K. Revathi, M. Babu, and R.Prabhakaran, Impact of dietary supplementation on the reproductive status of Japanese quail (Coturnix coturnix japonica) Thirunavukkarasu.N, D.Alexander, S.Muthu Kumar, P.Vijayanand and C.Sheeba Anitha Nesakumari, Marine crustacean bycatch resources and the production of chitin and chitosan from Royapuram fish landing center, Chennai P. Rajalakshmi, G.B. Brindha Devi, S. Mumtaj and S. Sheela, Effective vaccine development against Edwardsiella tarda P. Padmini, Contractual obligation vs social responsibility: teachers role in the development of concern for environmental protection AZRA Publications

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Foreword Food is of prime importance for a life to survive, sustain and grow, in addition to safer environment. With the burgeoning Indian/global human population, and shrinking resources like water, cultivable land and farm labourers etc., food security is currently the biggest challenge in the years to come. XV AZRA International Conference on “Recent Advances in Life Sciences” was an event organized by PG Departments of Zoology, Plant Biology and Plant Biotechnology and Biochemistry, Ethiraj College for Women, Chennai, Applied Zoologists Research Association (AZRA), Bhubaneswar and Zoological Survey of India, on 11-13 February, 2016 at Ethiraj College for Women, Chennai, Tamil Nadu. This conference was an event where issues related to production and productivity of the food from both animal and plant based resources and also the issues related to safer environment have been presented and discussed through 245 research papers in 12 different sessions of the conference. Above 300 applied biologists/ zoologists from different states of India and other countries like Botswana and Malaysia presented their research papers and discuss issues related to researches on the recent advances in life sciences. The main focus of this conference remained on researches related to food production system involving both animal and plant origin food products, leading to national and international food security with special reference to economic zoology, aquaculture, marine biology, veterinary and dairy sciences, plant protection, biotechnology and nanotechnology, phytochemistry, biochemistry and microbiology. Environment was another important issue covering biodiversity and forests, pollution and toxicology related to industrial wastes, agro-chemicals especially the pesticides and other aquatic as well as soil pollutants and their mitigation options to maintain the safer environment. I congratulate Dr. K. Revathi, Organizing Secretary and Co-Organizing Secretaries, Mrs. Prema Sampathkumar and Dr. Mrs. M. Sujatha of this Conference, Dr. Mrs. A. Nirmala, Principal and Secretary, Ethiraj College for Women, Chennai, and to all other faculty members of Ethiraj College for Women for their guidance and tireless efforts to make this event a grand success. I also congratulate Dr. Anand Prakash, General Secretary, AZRA and all members of AZRA family who participated in this conference from different states of India and other countries for the cause of life sciences and made significant contributors for this conference. I am hopeful that the recommendations of this conference will be useful to plan the future research and development programs/strategies in the field of life sciences especially to enhance the production and productivity of the food and protect the environment for mankind. Dr. B. Vasantharaj David, President AZRA, Chairman, Scientific and Academic Board Internationl Institute of Biotechnology & Toxicology Padappai- 601301, Tamil Nadu

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Proceeding and recommendations of XV AZRA International Conference on “Recent Advances in Life Sciences” Ethiraj Women College, Chennai, Tamil Nadu, 11-13 February, 2016 Population of the World is set to grow considerably and anticipated to exceed 9 billion by 2050. Food is the prime requirement of life, as source of energy to survive, sustain and multiply. Therefore, demand of the food will also be swelling to fulfill the requirement of this burgeoning global population. Food security either global and or national implies to make available the quality food with required quantity to every person at / reasonable price. Food, which includes food grains (cereals & pulses), edible oils from oil crops, fruits, vegetables, sugar, fish and fisheries products, eggs, milk and its products, meat etc., is the outcome of agricultural products from both plant as well as animal resources. Comparing to the global population dependency on agriculture sector of about 33%, above 60% of Indian population is dependent on agriculture, thus importance of the agriculture is foremost in India. Globally, India is foremost country in production of pulses, cotton fiber and jute and second to China in production of rice, wheat, vegetables, fruits and aquaculture. India ranks third in production of root and tuber crops and other cereals and ranks 6 th in production of edible/ vegetable oils. Growth in production of aquaculture in India is about 9% as compared to 7% per annum in the world and India ranks 4 th after China, Peru and Indonesia in total fish production. Of total 148 million tons fish production (capture fishery and aquaculture in the world, India contributes to about 10 million tons (FAO, 2015). With the burgeoning global human population, and shrinking resources like water, cultivable land and farm labourers etc., food security is the biggest challenge in the years to come. During the last five decades, there have been the major advances in food grains production in India due to the adoption of green revolution technology, which included high yielding varieties (HYV) and intensive use of chemical fertilizers and pesticides. Use of high yielding varieties, chemical fertilizers and pesticides have invited several new pest problems along with deterioration of soil health and environment, which are currently being added with climate change/ global warming. In major crops of agricultural importance, pests (insects, mites, nematodes etc.), diseases (fungal, bacterial and viral diseases) and weeds cause considerable yield losses (30-40%) every year in India. Climate change in terms of increased temperature, elevated CO 2 and erratic rainfall leading to draught, flood, submergence, and salinity situations, has direct and indirect effects on the outbreaks and epidemics of these pests. Therefore, pest management of the food and horticultural crops is one of the most important factors to be addressed, when one thinks of the food security. Agro-chemicals especially the pesticides (insecticides, weedicides, fungicides etc) used for plant protection are of great concern of environmental safety as these chemicals not only kill the target pests but also non-target bio-agents like natural enemies (parasites and predators), soil microbes, honey bees (pollinators of cross-pollinated crops), and adversely affect the biodiversity of the agro-ecosystems. Synthetic pesticides also contaminate the precious natural resources like soil and water, which directly or indirectly affect the aquatic life including aquaculture, more commonly during rainy season. Therefore, minimizing the use of pesticides is the global demand of the nature for safer environment. Currently attempts are being made to reduce the applications of synthetic pesticides either by developing biotic stress tolerant varieties or by using biopesticides (botanical/ plant origin pesticides, microbial, parasites and predators). Organic agriculture is one of such attempts being currently emphasized globally and more commonly in European countries, USA and Canada, where pest control is being emphasized to be done either using cultural practices or bio-pesticides or bio-control agents.

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Food production through animal resources like fish and fisheries, dairy and veterinary and also entomological resources like apiculture and entomophagy etc. is another important aspect of food security, which needs a due attention. Under present circumstances, global climate change resulting erratic rainfall, increase in temperature and carbon dioxide, reduction of water and land resources etc. is of great concern especially the water scarcity, which has direct impact on the food production through these resources. There is a need to develop technologies which need less water (per drop more crop) and other inputs or technologies to conserve/harvest the water through different means. There is a need of efficient fish/ animal breeding technologies having minimum inputs and quality and also high value inputs to cater the need of the food as well as income generation. XV AZRA International Conference on “Recent Advances in Life Sciences” was an event to celebrate glorious 27 years of Applied Zoologists Research Association (AZRA) on 11-13 February, 2016 at Ethiraj Women College, Chennai, Tamil Nadu. This conference was jointly organized by PG-Departments of Zoology, Plant Biology and Plant Biotechnology and Biochemistry, Ethiraj Women College, Chennai, Applied Zoologists Research Association (AZRA), Bhubaneswar (previously at CRRI, Cuttack) and Zoological Survey of India, Chennai. This conference has been sponsored by UGC, NBA, DST, DBT, ENVIS& ZSI. Organizing Committee of the conference including Patrons, Mr. M.V. Muralidharan, Chairman, Ethiraj Women College, & Dr. (Mrs.) A. Nirmala, Principal and Secretary, Ethiraj Women College, Chennai, Chairman, Dr. B. Vasantharaj David, President, AZRA and Co-Chairpersons, Dr. Anand Prakash, Founder & General Secretary, AZRA and Dr. R.W. Alexander Jesudasan, Vice President, AZRA and Principal & Secretary, Madras Christian College, Chennai & Dr. Kailash Chandra, Additional Director, Zoological Survey of India, Organizing Secretary, Dr. Mrs. K. Revathi, HOD, PG-Department of Zoology, Ethiraj College, Co-Organizing Secretaries, Mrs. Prema Sampathkumar, HOD, Department of Plant Biology and Plant Biotechnology and Dr. Mrs. M. Sujatha, HOD, Department of Biochemistry, Ethiraj Women College, Chennai was very active, meticulous and methodical to make this event a grand success. A galaxy of applied biologists/ zoologists from twenty one Indian states and abroad especially from Botswana and Malaysia presented their research papers and discuss issues related to researches on the recent advances in life sciences. Above 300 delegated registered for this conference, where 245 research papers were presented orally as well as posters in three concurrent sessions every day. The main focus of this conference remained on researches related to food production system involving both animal and plant origin food products, leading to national and international food security with special reference to economic zoology, aquaculture, marine biology, veterinary and dairy sciences, plant protection, biotechnology and nanotechnology, phytochemistry, biochemistry and microbiology. Environment was another important issue covering biodiversity and forests, pollution and toxicology related to industrial wastes, agrochemicals especially the pesticides and other aquatic as well as soil pollutants and their mitigation options to maintain the safer environment. The conference was inaugurated on 11 February, 2016 by Thiru M. V. Muralidharan, Chairman, Ethiraj Women College, Chennai, Dr. B. Vasantharaj David, President AZRA, Dr. (Mrs.) A. Nirmala, Principal and Secretary, Ethiraj Women College, Chennai, Sri T. Rabikumar, IFS, Secretary, NBA, Chennai, Prof. Balasubramaniam, Malayasia, Dr. Anand Prakash, Founder & General Secretary, AZRA and Dr. R.W. Alexander Jesudasan, Principal, Madras Christian College, Chennai. A key-note address was delivered by Col. Dr. C.P. Churamani on “Prospectives of biothreat and their mitigation strategies”, which was chaired by Prof. Mrs. B. Bharathalaxmi, Andhra University, Visakhapatanam. Further, Dr. S. S. Misra, Lucknow, Dr. Suresh Chanda, ZSI, Canning, W.B., Dr. S. K. Srivastava, Bhubaneswar, Prof. R.N. Singh, BHU, Varanasi, Dr. S.K. Shrivastava, IGKV, Rajanandagaon, CG, Dr P.V. Reddy, IIHR, Bangalore & Dr K. Sreedevi, IARI, New Delhi as jury members for the sessions of best oral and poster paper presentations for

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Prof. P. Kameswara Rao Award played a vital role to judge & select the young scientists for this award. Prof. Manendra Kumar, B. R. A. Bihar University, Muzaffarpur, Prof. P. Nagaraja Rao, Osmania University, Hyderabad, Prof. R.N. Ganguli from IGKV, Raipur and Profs B.K. Sahoo and L.K. Rath from OUAT, Bhubaneswar, Prof. P. Suresh from Madurai and many other delegates were very active for critically asking the questions to the presenters. A lead paper presented by Dr. Bishas Kar, Centre for Genetics and Research, The Madras Medical Mission, Chennai on “Genetic factors and etiopathogenesis of male infertility” was very attractive and informative. One of the important components of this event has been AZRA Awards Ceremony, wherein awards instituted by the Association of eight different categories for the years 2015 & 2016, selected by the AZRA Award Committee under the Chairperson, Dr. (Mrs.) Jagadiswari Rao, Founder and Vice President, AZRA, along with other committee members viz., Prof. S.K. Panda, OUAT, Bhubaneswar and Dr. J. K. Sundaray, CIFA, Bhubaneswar, were conferred to the researchers on 11 February, 2016 at Central Hall, Ethiraj Women College, Chennai. Chairpersons of different technical sessions viz., Dr V. Mohan & Dr N. Mathivanan (Plant Protection), Dr Babu Mano (Veterinary & Dairy Sciences), Dr. Niranjali Devaraj (Biotechnology & Nanotechnology), Dr P. Sundararaj & Dr A.G. Sreenivas (Economic Zoology), Dr M.S. Ramaswamy (Phytochemistry), Dr. D. Narsimhan (Biodiversity & Forestry), Dr Balasundaram (Biochemistry), Dr Geeta Vanage (Toxicology & Pollution), Dr Ganesan (Microbiology) and Dr Ajith Kumar (Aquaculture and Marine Biology) played a vital role for smoothly running the sessions and finally prepare the proceedings with the help of reppourters. During valedictory session, Dr. R. Prabhakaran, Former, V.C., T.N. Veterinary & Animal Sciences University, Chennai addressed the audience with his views about advances in veterinary and animal sciences researches in India. Dr. S.N. Sushil, Plant Protection Advisor, Government of India, Faridabad delivered a special lecture on current plant protection scenario in India, where he emphasized to minimize the use of synthetic pesticides to protect the environment by adopting non-pesticidal approaches viz., cultural practices, use of botanicals, & bio-control agents through conservation of natural enemies. Dr Anand Prakash, General Secretary, AZRA, lastly given concluding remarks about the proceedings of the conference, followed by vote of thanks by Dr. M. Sujatha, Co-organizing Secretary. I am grateful to all the faculty members of Ethiraj Women College, members of AZRA family and also the participants of this conference from different states of India and other countries who have joined this event for the cause of life science and remained very significant contributors for this conference. I owe my gratitude to Dr. B. Vasantharaj David, President, AZRA, Dr. Mrs. A. Nirmala, Principal and Sectrtary, Ethiraj Women College, Chennai, Dr. K. Revathi, Organizing Secretary and also Co-Organizing Secretaries, Mrs. Prema Sampathkumar, Dr. Mrs. M. Sujatha of this Conference for their guidance and tireless efforts to make this event a grand success. I am sure that excellent presentations and fruitful discussion during the 3 days deliberations might have enriched the participants with the knowledge and information on recent advances in life sciences. Recommendations from the conference deliberations & discussion Each living organism requires energy to survive, sustain and multiply and food is the only source of energy. Thus, food is of prime importance for a life, in addition to safer abode/ habitat/ surroundings, we call it an environment. XV AZRA International Conference on “Recent Advances in Life Sciences” was an event where issues related to production and productivity of the food from both animal and plant based resources and also the issues related to safer environment have been presented and discussed through 245 research presentations and the recommendations in brief are as follows:

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For the food production from animal based resources through aquaculture, captive fisheries and marine biology, there is a need to develop resource (water) efficient, economic and feasible production technology along with suitable post harvest management of the produce right from the production source to consumption. As water is becoming a scarce resource day by day due to failure of the monsoon and erratic climate, even water management needs to be linked with such technology development especially for aquaculture, because one cannot have aquaculture without sufficient quality fresh/brackish/marine water. To make this system sustainable and more profitable one can adopt Homestead Farming System (HFS) which has immense potential to meet several socio-economic and ecological conditions for poor households and contributes in their better living and sustainability rather than as a transformative livelihood activity. Impetus must be given for species and system diversification since cutting edge technologies has proven to be benefited the production and productivity by adopting diversification. Climate resilient aquaculture practice must be followed.

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Looking for alternate animal origin food sources, edible insects is another food source, as insects are consumed by human beings and termed as Entomophagy. Insects used for consumption are rich in protein, fats, vitamins and most essential micronutrients and also for medicinal values called as Entomotherapy. A number of insects including winged termites, grasshoppers, locusts, crickets, beetle grubs, ants and caterpillars are edible. In general insect larvae have high protein as compared to other protein sources for human beings. In the world, about 1400 insect species recorded in more than 370 genera and 90 families are being used for food, especially in central and southern Africa, Asia, Australia and Latin America. In India, several ethnic groups/ tribal people of north-eastern hill regions and eastern regions eat ants, termites, grasshoppers, larvae and pupae of bees, crane flies, wood boring beetle, dragon fly nymphs, queen termites, cockroaches, etc. There is a need to strengthen and popularize entomophagy, as insects can easily be mass multiplied due to their high reproductive potential and short life cycle.

3.

Plant based food production includes agriculture, which is a major source of food for mankind. Today, to enhance the food production & productivity through agriculture, there is need to develop or improve high yielding, climate resilient, short duration, photo-insensitive/ weakly photo-sensitive crop varieties having promising tolerance to abiotic (drought, salinity, submergence, temperature etc.) and biotic (pests and diseases) stresses.

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There is a need to study the crop-pest-natural enemy-weather relationship in important food crops (agricultural/ horticultural) under changing climatic conditions and to develop pest forewarning modules for specific key pests of agricultural importance, using both the historical and experimentally generated data. Due to shifts in crop cultivation practices as well as changing climate, crop pest scenario is being changed, therefore, it is important to work out/develop effective and pest surveillance procedures (visual, light trap, pheromone trap or e-pest surveillance) for timely pest management of the crops) and to intensively study the diversity of the pests as well as natural enemies in forest and agro-ecosystems.

5.

For plant protection, there is a need to develop location specific, holistic, economic, environmental friendly and effective IPM modules based on pest surveillance having need based application of quality pesticides with label claims. Action may be taken as soon as pest incidence reaches economic threshold level. IPM should be adopted in unified manner throughout the country through the central or state governments’ supervision. Today, farmers go with the recommendations of even pesticide sellers in the rural areas, who are misguiding the farmers.There is a need to develop effective and

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climate resistant strains of bio-control agents for accretive and inundative releases. Laboratory reared bio-control agents should be made more heat/temperature tolerant and virulent. 6.

Every year, post harvest losses of the agri-produce in India are 10-25% and there is no sufficient safe storage facilities in the country, therefore, it is essential to construct safe storage structures in the rural sector to avoid such losses.

7.

For the safer environment, there is a need to create awareness about the environmental pollution especially the aquatic pollution through industrial wastes and overuse of the pesticides and attempts should be made to minimize the use of toxicants causing pollution. There is a need to identify and conserve biodiversity in different ecosystems. Animal, fish and plant taxonomy needs to be given attention as there is an acute shortage of bio-taxonomists in India.

8.

India is rich in faunal and floral diversity, therefore, attempts should be made to isolate and identify the novel chemicals/ active components from these natural resources using biotechnological and biochemical approaches, which can be used for the management of pests and diseases, including diseases related to human health.

These recommendations may be useful to plan the future research and development programs/strategies in the field of life sciences especially to enhance the production and productivity of the food and protect the environment for mankind. Dr. Anand Prakash, Founder & General Secretary, AZRA, Managing Editor, Journal of Applied Zoological Researches, Co-Chairperson: XV AZRA International Conference, K-9B/285, Bhagabanpur, Patrapada, Bhubaneswar-751 019, Odisha, India Email: [email protected]

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Proc. XV AZRA International Conference “Recent Advances in Life Sciences”, Ethiraj College for Women, Chennai, 11-13 February, 2016

PROSPECTIVES OF BIOTHREATS AND THEIR MITIGATION STRATEGIES COL (DR.) C. P. CHURAMANI, DDRVS, HQ Dakshin Bharat Area, Chennai-600 009, Tamil Nadu ABSTRACT: No nuclear weapons are required to destroy or spread the fear of terrorism these days. The modern technique deadlier than weapons is this bio-terrorism. This is the cause of concern for every nation to counter this type of attack. For this, only measure which can be applied is to know about those diseases which can be spreaded easily and then take precautions to control or be away from them. Efforts should clearly be made for primary prevention of violence in any form and for secondary prevention and effective treatment when necessary. The strategies of bio- warfare mitigations include, sharply increased funding for public health infrastructures, which have been starved for funds for years, should be provided at every level; effective surveillance for disease outbreaks, whatever their origin, is essential; training for public health and medical personnel in handling emergencies, whatever their cause, should be expanded, as should access to public health and medical services, without financial or other barriers. Earlier detection through periodic testing would allow for timelier implementation of response strategies such as vaccination, slaughter, disposal, cleaning and disinfection. Hence, by decreasing the time of unobstructed disease spread, screening could reduce economic damages. The people should be educated on ways to avoid and respond to health problems of all types. If a bioterrorism event did not occur, it would be useful to strengthen medical and public health infrastructures so they can respond effectively to other health emergences- also called dual-use strategy. Key words: Biothreat, bio-terrorism, bio-weapons, Anthrax, Bt agents,

INTRODUCTION Bio-terrorism (BT) can be defined as the use of microorganisms with intent of causing infection in order to achieve certain goals. With increased availability of biological agents and the technical information to produce them, bio-terrorism may become the weapon of choice in future. The implication for public health is that events of infection of a number of people is a cause of concern and carry different responsibilities for municipal, provincial and Government departments (Arora et al., 2002). The attraction for bio-weapons (BWs) is attributed to their followingfeatures: Low production costs - BWs are aptly called the “Poor Man's Atomic Bomb (Atlas, 1998) “Poor Man's Weapons of Mass Destruction (Batra, 2000). For atomic bombs, conventional weapons and nerve-gas weapons, the cost per casualty would be approximately $2000, $800 and $600, respectively. However, for BWs the cost would be about $1 per casualty (Atlas, 1998). Large quantities can, in most cases, be produced in a short period (a few days to a few weeks) at small facilities scattered over a large area (Batra, 2000). Non-detection by routine security systems (Batra, 2000) and easy access to a wide range of disease-producing biological agents (Whitby and Rogers, 1997) are other attractions. Biological toxins are among the most toxic agents known e.g. the quantity of botox in the dot of an 'i' is , when delivered properly, enough to kill ~10 people (Batra, 2000). A single microbial bio-weapon can, because it reproduces in the host, theoretically produce the desired detrimental outcome in a target host (Batra, 2000). BWs have the added advantage of destroying an enemy while leaving his infrastructure intact as booty for the winner (Batra, 2000).

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Bio-terrorism agents/biological agents (Table-1) can be used to spread infection through the air, water or through food. As in the past few years there have been various terrorist attacks in the world. India has been and still is a target for terrorist attacks. Therefore, it has become important for our country to remain alert especially in the field of public health. Table-1: List of potential bio-terrorism agents Category A (most likely to be used as BT agents)

Category B (second most likely to be used as BT agents)

Category C (third most likely to be used as BT agents)

Bacillus anthracis (anthrax)

Coxiella burnetti (Q fever)

Nipah virus

Clostridium botulinum toxin (botulism)

Brucella species (brucellosis)

Hantaviruses

Yersinia pestis (plague)

Burkholderia mallei (glanders) Tickborne hemorrhagic fever viruses

Variola major (smallpox)

Ricin toxin from Ricinus communis (castor beans)

Tickborne encephalitis viruses

Francisella tularensis (tularemia)

Epsilon toxin from Clostridium perfringens

Yellow fever

Viral hemorrhagic fever

Staphylococcus enterotoxin B (SEB)

Multi-drug resistant tuberculosis

(Source: Centers for Disease Control and Prevention, 2001) Characteristics of a perfect BW  Highly infectious and highly effective  Efficiently dispersible  Readily grown and produced in large quantities  Stable on storage  Resistant enough to environmental conditions  Resistant to treatment Disadvantages of BW  Difficulty of protecting the workers at all stages of production, transportation, loading of delivery systems and final delivery.  Difficulty in maintaining quality control and sufficient containment during growth and harvesting of agents.  Effective delivery problems: Most biological materials, including spores, are destroyed by exposure to UV light and drying. Agents released in the air may disperse in unexpected ways due to the vulgarities of wind patterns.  Poor storage survival: Many BWs must be stored under special conditions to maintain efficacy. Further, they are often difficult to maintain in a weapons - delivery state (e.g. loaded and ready to be fired in a rocket).  Difficult to control once released. Economic effect of biowarfare The economic implications of bio-warfare and their mitigation options has become a more pertinent issue as the fears of agricultural terrorism have grown. From an economic perspective, agricultural terrorism would cause damages by disrupting agricultural commodity and related markets either because of the events themselves or because of potentially expensive and intrusive

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mitigation actions. It has been documented that even unintentional outbreaks of diseases such as Foot and Mouth Disease (FMD) as potential bioterrorism agentscan cause substantial economic damages (Thompson et al., 2003). The total cost of a disease outbreak policy is composed of post outbreak economic losses brought by a disease outbreak weighted by outbreak probability plus the cost of any pre outbreak actions. This, in most cases, inherently implies a tradeoff between pre outbreak prevention and detection related costs & ex-post losses from the outbreak management plus any associated recovery costs. Analysis of prevention and response strategies directed toward Foot and Mouth Disease (FMD) have been the topics of numerous studies (Bates et al., 2003a,b; Garner and Lack, 1995; Schoenbaum and Disney, 2003; Berentsen et al., 1993). All of these studies mainly concentrate on post outbreak disease management including the optimal use of vaccination and slaughter. Less attention has been devoted to pre event surveillance and detection systems, which could allow for timely and more effective response measures but costs money whether or not an outbreak ever occurs. Although some attention has been raised towards surveillance systems (Bates et al., 2003b; Akthar and White, 2003), no empirical investigation has been performed, to the best of our knowledge, on the merit of such policies relative to vaccination and slaughter. Current US programs to detect and prevent FMD place a great deal of reliance on the recognition and reporting of clinical signs by producers, animal care takers, meat inspectors or veterinarians (Bates et al., 2003b). Reliance on such an approach raises two major problems. First, detection based on visual observation of clinical signs implies that the disease could have been present and possibly spreading before the realization of its presence. Second, clinical signs of FMD are indistinguishable from the signs of other diseases (Bates et al., 2003c,d). Therefore, more reliable methods for detection of FMD may be appropriate. One of the possible surveillance and detection systems that could be used involves the use of periodic screenings of animals. Such screenings could detect FMD carriers before clinical signs appear. Earlier detection through periodic testing would allow for timelier implementation of response strategies such as vaccination, slaughter, disposal, cleaning and disinfection. Hence, by decreasing the time of unobstructed disease spread screening could reduce economic damages. In general, how quickly a person receives a correct diagnosis depends upon what symptoms are evident, how astute the treating physician is, and what access the treating facility has to advanced diagnostics. For example, Anthrax’s initial presentation can closely resemble that of common respiratory viruses, making the disease difficult to distinguish from flu-like illness (Bell et al., 2002). Terrorists groups undertake this activity in a smaller or larger scale than the rival countries. Basically the bio-agents used are the same as in biological warfare. Bioterrorism is an attractive weapon because biological agents are relatively easy and inexpensive to obtain, can be easily disseminated, and can cause widespread fear and panic beyond the actual physical damage they can cause. Zoonotic nature of biowarfare agents Many modern diseases, including diseases that became epidemics, started out as zoonotic diseases and have animal reservoirs. Epidemic and pandemic diseases often depend on a constant influx of humans who have not developed an immune response, so in the past tended to burn out after their first run through a population. In all of human infectious diseases 60% are zoonotic and 75% of emerging human infectious diseases have an animal disease origin. In most diseases, biological pathogens have to be a chronic infection in order to stay alive in the host for long periods or have a non-human to live in while waiting for a new host. Major factors contributing to the appearance of emerging zoonotic pathogens in humans are drastically increased when humans encroach into areas of wildlife or the movement of wild animals into area of human activity. One case of note was the bubonic plague. The plague caused an estimated 200 million deaths throughout Europe. Densely packed cities provide ample breeding ground for diseases and many hosts to infect, while

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the infection of a single farm has the potential to affect thousands or millions of people. Zoonoses like bovine encephalitis, or Mad Cow disease, have the ability to cause an entire country's farming exports to shut down (WHO, 2010). Zoonotic diseases occur primarily in areas that involve the handling of livestock, both on large country farms and in smaller, urban settings, such as rooftop gardens that raise chickens. These human-animal interactions provide the base for most outbreaks of zoonoses. Those in poorer communities are also less likely to be correctly diagnosed as having contracted new diseases and therefore, often are not correctly treated. Unsurprisingly, a lower priority is given to control zoonotic diseases and the high cost of diagnosing and treating those people mean the outcomes for those communities is particularly bleak. Of the 282 deaths from avian influenza since 2003, the overwhelming majority have been from developing countries (Anonymous, 2009). In developing countries, the deterioration of veterinary services and transferring to the private sector mean that people in those areas are less likely to be able to invest in treatment or preventive measures for their livestock, which result in animals with poor health. The consumers in the developing world are also most likely to be purchasing cheaper meat and milk from outlets where it is not inspected or refrigerated properly, or in the case of milk, pasteurized, allowing the spread of diseases that can be passed through consumption of animals. Mitigation strategies against biowarfare Zoonotic diseases impose a disproportionately high burden on the developing world. Controlling the spread is nearly impossible, and yet there are many ways of preventing the sort of rapid spread that would make for a global disaster. In many cases, the control measures which have worked well in Europe cannot easily be applied in developing countries. For example, in the United Kingdom, brucellosis, a flu-like disease, was eradicated entirely, and killing entire herds of infected cattle controls bovine tuberculosis before it can spread, or infect humans. This approach would not be feasible where people depend on their animals for their livelihoods and replacements are hard to obtain. However, public awareness campaigns such as the importance of boiling milk could be undertaken but rarely implemented. Health policy makers are usually unaware of the consequences imposed by these diseases. Multiple mitigation strategies can be applied for BioWarfare. A. Research is needed to establish where these diseases are present, identify the risk factors, which make particular groups of people or livestock likely to contract them and find cost effective ways of dealing with them. A change in policy and attitudes is also needed to the control of the neglected diseases. B. Most priority bioterrorism agents are zoonotic in origin. Moreover, for a number of biological terrorism agents, there is evidence that animals could provide early warning of an acute attack. As a result, an attack on human populations with a bioterrorism agent would likely pose a health risk to animal populations in the target area; therefore, integrating veterinary and human public health surveillance efforts is essential. The animals can be used as "sentinels" of a human bioterrorism attack. Therefore, integrating veterinary and human public health surveillance efforts is essential. The goal should be "prompt diagnosis of unusual or suspicious health problems in animals," as well as establishing criteria for investigating and evaluating suspicious clusters of human and animal disease or injury and triggers for notifying law enforcement of suspected acts of biological terrorism. Veterinarians play an important role in such scenarios, as they are more coherent in such diseases vis-a-vis physicians who have seldom encountered such cases. Disease forecasting using sentinels, reporting by faster diagnostic technique, undertaking prompt treatment, implementing control measures and planning prevention constitute the veterinary public health measures. Therefore, a well equipped veterinary laboratory with adequate technical expertise is well suited to undertake diagnostic investigations in case of any bioterrorism

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attack. Certain characteristics of an outbreak in animals might help differentiate a terrorist event from a natural event: i. Increased morbidity or mortality. ii. Appearance in a species not normally manifesting the disease. iii. Occurrence at the wrong time or an unusual time of year. iv. Occurrence in areas where it is not normally found. v. Lack of response to the normal mode of treatment. Table- 2: Bacterial and Viral Threat Agents Associated with Animals Category A Diseases and Zoonotic Category B Diseases and Zoonotic Agents Agents Foot-and-mouth disease No Brucellosis (Brucella spp.) Yes (Picornaaphtho virus) Anthrax (Bacillus anthracis) Yes Epsilon toxin of Clostridium No perfringens Botulism (Clostridium No Food safety threats Yes botulinum toxin) (Salmonella spp., Escherichia coli O157:H7, Shigella) Plague (Yersinia pestis) Yes Glanders (Burkholderia Yes mallei) Smallpox (Variola major) No Melioidosis (Burkholderia Yes pseudomallei) Tularemia (Francisella Yes Psittacosis Yes tularensis) (Chlamydiapsittaci) Viral hemorrhagic fevers No Q fever (Coxiella burnetii) Yes (Ebola, Marburg, Lassa, Machupo viruses) Ricin toxin No Staphylococcalenterotoxin B No Typhus fever (Rikettsia No prowazekii) Viral encephalitis (VEE, Yes EEE, and WEE viruses) Water threats (Vibrio Yes cholerae, Cryptosporidium parvum) (Source: NRC, 2002a, 2003a) C.

Clustering of cases, or some other information leading to suspicion of an attack, days could elapse before the first cases were correctly diagnosed. Once even a few cases (perhaps even one) were definitly shown to result from bio-agent exposure, however, actions would be initiated to determine the probable origin and to establish whether the exposure was of natural or man-made cause.

D.

Counter bioterrorism measures include identification of infectious sources, surveillance system, disease reporting system, early detection and management of a biological terrorism attack. As the popular saying to be forewarned is to be forearmed, given advance notice, even by weeks, of an impending BT outbreak, the hope exists that the tools and imaginations of molecular biology will find the means to prepare some effective biological defense (Centers for Disease Control and Prevention, 2001). The private sector has little

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RECENT ADVANCES IN LIFE SCIENCES :Proc. of XV AZRA Inter. Conf., 2016 incentive to develop diagnostics for Bio-Warfare agents (as they are considered rare diseases) and the civilian sector has neither the charter nor coordination to implement a wide-scale surveillance-based information-technology network. One strategy would be to assess the feasibility of developing rapid, broad-spectrum diagnostic tools for detecting and identifying pathogenic microorganisms. Indeed, because emerging diagnostic technologies like gene chips (DNA microarrays) and protein microarrays offer a way to provide thousands of concurrent assays in a single test format, it seemed reasonable to assume that investments in cost reduction or readout simplification could lead to practical diagnostic devices with the desired characteristics. The diagnostic technology would also offer direct patient benefit, hastening its acceptance by the medical community (Szpiro et al., 2007). Table-3: Recommended BSL for BT agents Agent

Bio Safety Level (BSL) Specimen Handling 2 2

Culture Handling 3 3

Brucellaspp.

2

3

Burkholderia pseudomallei Burkholderia mallei Coxiella burnetii Clostridium botulinum

2 2 2 2

3 3 3 3

Alphaviruses Bacillus anthracis

Francisella tularensis Yersinia pestis Smallpox Staphylococcalenterotoxin B VHF

E.

2 3 2 3 4 4 2 2 4 4 (Source: Anonymous, 2006) Training of all stake holders at different technical level. Providing of training module for mass panic management to E-learning, awareness generation and knowledge management for bio-defence services, para military and civilian defence staff.

Conclusion: Biological terrorism is the threatened use or use of a microorganism or toxin derived from living organisms to induce death or disease in people, animals or plants. Nearly all of the potential bioterrorism agents are zoonotic in nature. Zoonotic diseases impose a disproportionately high burden on the developing world. Controlling the spread is nearly impossible, and yet there are many ways of preventing the sort of rapid spread that would make for a global disaster. Efforts should clearly be made for primary prevention of violence in any form and for secondary prevention and effective treatment when necessary. The strategies of bio- warfare mitigations includes, sharply increased funding for public health infrastructures, which have been starved for funds for years, should be provided at every level; effective surveillance for disease outbreaks, whatever their origin, is essential; training for public health and medical personnel in handling emergencies, whatever their cause, should be expanded, as should access to public health and medical services, without financial or other barriers. Earlier detection through periodic testing would allow for timelier implementation of response strategies such as vaccination, slaughter, disposal, cleaning and disinfection. As the popular saying to be forewarned is to be forearmed, given advance notice, even by weeks, of an impending BT outbreak, the hope exists that the tools and imaginations of molecular biology will find the means to prepare some effective biological defense.

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REFERENCES Akhtar, S. and White, F. 2003. Animal Disease Surveillance: Prospects for Development in Pakistan, Scientific & Technical Review Intern. Office of Epizootics.22(3): 977-987. Anonymous, 2006. Sentinel level clinical microbiology laboratory guidelines for suspected agents of bioterrorism and emerging infectious diseases. Clinical Laboratory Bioterrorism Readiness Plan,American Society For Microbiology, pp ???. Anonymous, 2009. Cumulative Number of Confirmed Human Cases of Avian Influenza A/(H5N1), Reported to WHO, http://www.who.int/csr/disease/ avian_influenza/ country/cases_table_2009_12_30/en/index.html. Arora, D. R., Gautam, V. and Arora, B. 2002. Biological warfare: Bioterrorism. Indian J Med Microbiol.20: 6-11. Atlas, R.M. 1998. The Medical threat of biological weapons.Critical Reviews Microbiol.24(3): 157-168. Bates, W. T., Carpenter, T. E., Thurmond, M .C. 2003a.Benefit-Cost Analysis of Vaccination and Preemptive Slaughter as a Means or Eradicating Foot-and-mouth Disease, American Journal of Veterinary Research, 64: 805-812. Bates, T. W., Thurmond, M. C., Hietala, S. K., Venkatesvaran, K. S., Wilson, T. M., Colston, Jr. B. W., Trebes, J. E., and Milanovich, F. P. 2003b. Surveillance for Detection of Foot-and-Mouth Disease.Commentary in Journal of the American Veterinary Medical Association,223(September 2003): 609-614. Bates, T. W., Thurmond, M. C. and Carpenter, T. E. 2003c. Description of an Epidemic Simulation Model for use in Evaluating Strategies to Control an Outbreak of Foot-and-Mouth Disease, American Journal of Veterinary Research.64: 195-204. Bates, W. T., Thurmond, M. C. and Carpenter, T.E. 2003d. Results of Epidemic Simulation Modeling to Evaluate Strategies to Control an Outbreak of Foot-and-Mouth Disease,American Journal of Veterinary Research, 64(February 2003 b): 205-210. Batra, H.V. 2000. International response to BW threat and the global scenario.In: XXIV National Congress of Indian Association of Medical Microbiologists. Patil CS (ed) (Department of Microbiology, JNMC, Belgaum) 2000. Bell, D. M., Kozarsky, P. E. and Stephens, D. S. 2002. Clinical Issues in the Prophylaxis, Diagnosis, and Treatment of Anthrax, Emerging Infectious Diseases, 8(2): 222–225. Berentsen, P. B. M., Dijkuizen, A. A. and Oskam, A. J. 1992. A Dynamic Model for Cost-Benefit Analysis of Foot-and Mouth Disease Control Strategies.Preventive Veterinary Medicine.12 (March, 1992): 229-243. Garner, M. G. and Lack, M. B. 1995. An Evaluation of Alternative Control Strategies for Foot-and-Mouth Disease in Australia: A Regional Approach.Preventative Veterinary Medicine.23(May, 1995): 923. Schoenbaum, M. A. and Disney, W. T. 2003.Modeling Alternative Mitigation Strategies for a Hypothetical Outbreak of Foot-and-Mouth Disease in the United States.Preventative Veterinary Medicine, 58(April, 2003):25-52. Szpiro, A., Johnson, B. and Buckeridge, D. 2007. Health Surveillance And Diagnosis For Mitigating A Bioterror Attack, Lincoln Laboratory Journal17 (1): pp.96-107. Thompson, D., Muriel, P., Russell, D., Osborne, P., Bromley, A., Rowland, M., Creigh-Tyte, S., and Brown, C. 2003.Economic Costs of Foot and Mouth Disease Outbreak in United Kingdom in 2001.”Department for Environmental, Food and Rural Affairs, UK. 2003. [On-line] Available at: http://www.defra.gov.uk/ corporate/inquiries/lessons/ fmdeconcostrev.pdf Last Accessed 10/06/04. Whitby, S. and Rogers, P. 1997.Anticrop biological warfare - implications of the Iraqi and U.S. programs.Defence analysis13:303-318. WHO, 2010.Global Classrooms.Lebanon Model UN Conference.

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Proc. XV AZRA International Conference “Recent Advances in Life Sciences”, Ethiraj College for Women, Chennai, 11-13 February, 2016

INCIDENCE AND ANTIMICROBIAL SUSCEPTIBILITY TESTING OF LISTERIA MONOCYTOGENES IN FOUR STREET-FOOD VENDING SITES IN GABORONE, BOTSWANA D. LOETO1, E. MONTHUSI2, B. LETSHOLO3, and K. WALE1 ABSTRACT: Listeria monocytogenes is widely distributed in nature and a significant foodborne pathogen. Where outbreaks of listeriosis have occurred, high fatalities ensue especially among the immune suppressed, pregnant women and their fetuses. The current study investigated the occurrence of the L. monocytogenes in various foods sold by street vendors in four geographical areas of Gaborone from October 2011 to March 2012. From a total of 396 ready-to-eat street foods cultured, 60 (15.2%) tested positive for the organism. Out of the 60 confirmed isolates of Listeria monocytogenes, 48 (12.1%), 6 (1.5%) and 6 (1.5%) were isolated from vegetable, protein and starch food portions, respectively. From the four geographical areas selected for sampling in this study, the UB area recorded the highest number 24 (6.1%) of positive isolates while the bus station area recorded the least, 6 (1.5%). Thirty per cent of the positive isolates were resistant to tetracycline, 10% were resistant streptomycin, chloramphenicol and erythromycin each. The outcomes of the present investigation revealed the presence of L. monocytogenes in foods sold by street food vendors in Gaborone. This suggests a need for vigilance in implementing food hygiene training programs in the country. Key words: Listeria monocytogenes, antibiotic resistance, listeriosis, street foods

INTRODUCTION The gram positive and facultative intracellular microorganism, Listeria monocytogenes although less common in cases of sporadic food-borne infections, remains important in the overall burden of food-borne disease.For example, in the United States of America it is said to account for 4% of all hospitalizations and 28% of all deaths from food-borne disease (Mead et al., 2006). The elderly, newborns, pregnant women and the immune suppressed are thought to be the most susceptible to listeriosis. Symptoms of the disease include meningoencephalitis, septicemia, gastroenteritis and abortion as a result of prenatal infection (WHO, 2002). A wide assortment of foods such as meat and meat products, milk and milk products, seafood and salads has been implicated in food-borne listeriosis (Jebelli et al., 2012; Kawasaki et al., 2009). Also, ready-to-eat foods (RTF) may serve as important vehicles in the transmission of the pathogen (Osaili et al., 2011). L. monocytogenes is known to thrive under refrigeration conditions and it can survive well in low pH foods such as yoghurt (Gahan et al., 1996) and is also capable of growth on dry surfaces (Wong, 1998). These characteristics particularly make the organism to be well adapted to colonize the food environment. Ever since the first report of antibiotic resistant L. monocytogenes in 1988 (Poyart-Salmeron et al., 1998) resistance of the pathogen to antibiotics has become a significant worldwide public health concern (Kovacevic et al., 2013). Infections by Listeria are routinely treated by β-lactam antibiotics such as penicillin and ampicillin but in immunocompromised individuals, the treatment regime is augmented with aminoglycosides like trimethoprim (Hof, 2003). Although L. monocytogenes has previously been detected in various food products retailing in Botswana (Manani et al., 2006; Letsholo et al., 2008), little data exists on the occurrence of the pathogen in street vended foods. This therefore, necessitates further investigation of this organism 1

Department of Biological Sciences, University of Botswana, Gaborone, Botswana Department of Environmental Health, Mahalapye Sub-District, Mahalapye, Botswana. 3 Department of Environmental Health, University of Botswana, Gaborone, Botswana. Corresponding author e-mail: [email protected] 2

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as well as the disease caused by this bacterium. The aim of the present study was to follow up Morobe et al., (2009) study and assess the susceptibility of 68 isolates of L. monocytogenes isolated in street vended foods in order to monitor of its prevalence and antibiotic resistance dynamics.

MATERIALS AND METHODS Sampling was done from October 2011 to March 2012. A plate of food was bought randomly from street food vendors in four different localities of Gaborone (BBS mall, University of Botswana (UB), Bus Station, and Bus Rank). The plate consisted of starch, protein and vegetable/salad portions. To prevent cross contamination, each plate was placed in a separate properly labeled sampling bag (Whirlpak, Nasco, Fort Atkinson, Wiscosin, USA). The plates were then immediately transported to the lab in a cooler box containing ice packs. Each locality was sampled at least twice during the course of the study. Upon arrival at the lab, a 10g of each portion of the three portions on one plate (that is starch, protein and salad/vegetable) was weighed and transferred to a sterile stomacher bag containing 90ml Listeria enrichment broth (Oxoid, Basingstoke, UK) and then homogenized using the stomacher (Seward 400, Tekmar, Cincinnati, Ohio, USA) set at medium speed. The homogenate was then transferred to a sterile conical flask, covered and then allowed to shake at 90rpm on an incubator shaker (Innova 4000, New Brunswick Scientific, Edison, New Jersey, USA) set at 37ºC for 24h. A 0.1ml of the overnight culture was aseptically spread plated on modified listeria selective agar (Oxoid) supplemented with listeria selective supplement (Oxoid) and subsequently incubated at 37ºC for 24h. Typical Listeria colonies appearing dark brown with black zones were sub-cultured on tryptone soy agar (Merck, Darmstadt, Germany) and incubated at 37ºC. Gram staining was performed on the colonies and Gram positive short rods were further grown on sheep blood agar. Isolates that displayed β-hemolytic activity on sheep blood agar were sub-cultured on tryptone soy agar (Merck) slants and incubated at 37ºC for 24h and thereafter maintained at 4ºC. The strains on slants were then identified using the API Listeria identification kit (bioMeriéux, Marcy l’Etoile, France) according to manufacturer’s instructions. Positive isolates were further confirmed by the VITEK 2 (bioMerieux, Hazelwood, Missouri, USA) automated identification system according to the instructions from the manufacturer. This identification system has higher discriminatory power because it identifies bacteria based on 64 biochemical substrates compared to API Listeria which has 20 substrates. Positive L. monocytogenes isolates were preserved at -80ºC in tryptose soy broth (Merck, Darmstadt, Germany) containing 20% glycerol and the preserved isolates were used in antimicrobial susceptibility testing. Antimicrobial susceptibility testing was performed on all the confirmed L. monocytogenes isolates. The isolates were first standardized by growing them on Mueller-Hinton broth (Oxoid) for 24h and then the turbidity of the physiologically active culture was adjusted with sterile saline solution to obtain optical density comparable to 0.5 McFarland standards. One millimeter of the standardized suspension was then spread evenly on the surface of MuellerHinton agar (Oxoid) using a sterile bent glass rod. The susceptibilities of all isolates to different antibiotics were tested by the disk-agar diffusion method using criteria set by the CLINICAL LABORATORY STANDARDS INSTITUTE (CLSI) (2006). The following antimicrobial disks and concentrations were used for susceptibility testing; chloramphenicol (25 μg), erythromycin (5 μg), fusidic acid (10 μg), methicillin (10 μg), novobicin (5 μg), penicillin G (1 U), streptomycin (10 μg), tetracycline (25 μg) (Mast Diagnostics, Merseyside, UK). Listeria monocytogenes ATCC 19115 was used as the reference strain. The obtained data was analyzed using the Statistical Package for Social Sciences (SPSS 12.0, SPSS, Chicago, Illinois). Pearson’s correlation coefficient was used to determine differences in means among street vended foods obtained in four geographical areas of Gaborone, Botswana (P=0.01).

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RESULTS AND DISCUSSION In this study, from a total of 396 street-vended food samples examined, 60 (15.2%) tested positive for Listeria monocytogenes. The organism was detected in all the three food products analyzed, albeit with varying rates. Table 1 shows that out of the 60 positive isolates, salad/vegetable portion recorded the highest incidence (80%) of Listeria monocytogenes, whilst both protein and starch parts recorded 10% each. The prevalence of the organism among the foods tested was significantly different (p