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Advances in Animal Sciences and Biomedicine in 21st Century

Editors: Kuldeep Dhama, Yashpal Singh Malik, Muhammad Munir, K Karthik, Ruchi Tiwari and Sunil K Joshi

IAB

International Academy of Biosciences

International Academy of Biosciences (IAB) (http://iab-uk.org/)

Advances in Animal Sciences and Biomedicine in 21st Century

Editors Kuldeep Dhama Yashpal Singh Malik Muhammad Munir K Karthik Ruchi Tiwari Sunil K Joshi    

International Academy of Biosciences (IAB) (http://iab-uk.org/)

1st International Satellite Symposium (ISS)–2016, held on 12th October 2016 of International Academy of Biosciences (IAB) on

Advances in Animal Sciences and Biomedicine in 21st Century     EDITORS: Kuldeep Dhama Principal Scientist, Division of Pathology, ICAR-Indian Veterinary Research Institute, Izatnagar-243 122, Bareilly (UP), India

Yashpal Singh Malik Principal Scientist & National Fellow-ICAR, Division of Standardization, ICAR-Indian Veterinary Research Institute, Izatnagar-243 122, Bareilly (UP), India

Muhammad Munir Avian Viral Diseases Programme Compton Laboratory, Newbury, Berkshire, RG207NN, UK

K Karthik Assistant Professor, Central University Laboratory, Tamil Nadu Veterinary and Animal Sciences University, Madhavaram Milk Colony, Chennai, India

Ruchi Tiwari Assistant Professor, Department of Veterinary Microbiology, College of Veterinary Sciences, DUVASU, Mathura, Uttar Pradesh, India

Sunil K Joshi Laboratory Head, Cellular Immunology, College of Health, Sciences, Old Dominion University, Norfolk, VA 23508 USA

The views expressed in the articles including the contents are sole responsibility of the respective authors. The editors bear no responsibility with regards to source, and authenticity of the contents.

Correct Citation Dhama, K., Malik, Y.S., Munir, M., Karthik, K., Tiwari, R. and Joshi, S.K. (eds). 2016. Advances in Animal Sciences and Biomedicine in 21st Century. International Academy of Biosciences (IAB), p. 210

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About International Academy of Biosciences (IAB)

I

nternational Academy of Biosciences (IAB) (http://iab-uk.org/) is an autonomous multi-disciplinary research and educational organization registered in England and Wales, United Kingdom (UK) on 13 June 2016 under the registration number 10228061. Academy publishes international peer reviewed research journals in several areas of science (http://iab-uk.org/research/). The academy is mainly engaged to improve and upgrade science as well as research culture within developing countries and international corresponding institutes. The academy recognizes excellent researchers from all over the world with various IAB awards, honors and recognitions and further provides grants/scholarship, and travel grants for high achievements in the scientific field as well as to support the professional development of early-career & deserving scientists and researchers. Academy organizes seminars, conferences, workshops, trainings (HRD) on different areas of science in different countries for promoting science.

CONTENTS 1.

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Combating antimicrobial resistance: a drive towards better performance- an overview Hafiz M.N. Iqbal Fresh water blues: “brain-eating amoeba” in Indian subcontinent Sunil K. Joshi Genetics and vaccine efficacy of wild birds origin avian paramyxoviruses in domestic chickens Muhammad Zubair Shabbir, Sameera Akhtar, Yi Tang,Tahir Yaqub, Arfan Ahmad, Ghulam Mustafa, Muhammad Azhar Alam and Muhammad Munir Bluetongue in India: outbreaks to vaccine outcome Minakshi Prasad, Koushlesh Ranjan and Gaya Prasad Brief overview: bacterial diseases of equines S.K. Khurana and Kuldeep Dhama Vaccine approach for the ‘therapeutic management’ of incurable Mycobacterium avium subspecies paratuberculosis infection in domestic livestock population Saurabh Gupta, ShoorVir Singh, Kundan Kumar Chaubey, Manju Singh, Ashok Kumar Bhatia and Jagdip Singh Sohal Role of inflammation in severity of viral disease with focus on Herpes Papilloma virus Maryam Dadar and Kuldeep Dhama AIDS: spread the KNOWLEDGE… not the VIRUS Pranveer Singh OIE recommended nucleic acid based techniques for poultry diseases diagnosis Manas Ranjan Prabhraj, Kuldeep Dhama and Deepak Kumar Brucellosis- a veterinary perspective Himani Dhanze, R.S. Rathore, M. Suman Kumar, K. Dhama and Ashok Kumar Medicinal plants: emerging strategies for gut health, immunity and its development in poultry Jai Sunder, Sujatha Tamilvanan and Anandamoy Kundu Influence of some natural feed additives on performance, serum biochemical parameters and oxidative status in laying hens Mahmoud Alagawany, Mohamed Ezzat Abd El-Hack, Mayada Ragab Farag and Kuldeep Dhama Physiological interventions for addressing reproductive dysfunctions in broiler breeder hens J.S. Tyagi, Moudgal R.P., Gautham Kolluri and Gopi M. Tendon tissue engineering: latest trends and future perspectives Rekha Pathak Carrier state in domestic and wildlife species: a debatable issue of concern in the epidemiology of foot-and-mouth disease M. Rout Animal enteric viral emergencies: An overview Yashpal S. Malik, Sharad Saurabh, Jobin J. Kattoor, Shubhankar Sircar, Pallavi Deol and Kuldeep Dhama

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19. 20. 21. 22.

Comparison of concepts in treating bovine mastitis for developing better regime R.B. Rai, K. Dhama, M. Saminathan and S.S. Hingade Swinepox- epidemiology, pathogenesis, diagnosis and the control strategies Deenanath Ashokkumar, Nikunj Gupta, Awadh B. Pandey and Muthannan Andavar Ramakrishnan Development of designer vaccines using reverse genetics technology C. Madhan Mohan, Sohini Dey and Dinesh Chandra Pathak Contagious agalactia: a probable threat for small ruminants Rajneesh Rana Ochratoxicosis in poultry Anju Nayak, Sunil Nayak and Varsha Sharma Persistence of foot-and-mouth disease virus Rajeev Ranjan, Jitendra Kumar Biswal and B. Pattnaik

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ABSTRACTS 1.

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Development of real time PCR assay for the detection and absolute quantification of Mycoplasma bovigenitalium in large ruminants S. Behera, R. Rana, P.K. Gupta, Sonal, D. Kumar, Rekha V., Arun T.R., and D. Jena Bio-incidence of Mycobacterium avium subspecies paratuberculosis infection in suspected domestic livestock population in India using multiple tests Kundan Kumar Chaubey, Sahzad, Shoor Vir Singh, Premanshu Dandapat, Saurabh Gupta, Bjorn John Stephen, Manju Singh, Jagdip Singh Sohal, Mukta Jain, Gajendra Kumar Aseri, Manali Dutta, Neelam Jain, Parul Yadav, Jitendra Chauhan and Kuldeep Dhama Evaluation of recombinant CFPs based cocktail ELISA vis-à-vis Indigenous ELISA using semi-purified protoplasmic antigen to differentiate infected from vaccinated population of domestic livestock Kundan Kumar Chaubey, Rinkoo Devi Gupta, Saurabh Gupta, Shoor Vir Singh, Ashok Kumar Bhatia, Sujata Jayaraman, Naveen Kumar, Abhishek Singh Rathore, Sahzad, Bjorn John Stephen, Manju Singh, Mukta Jain, Kuldeep Dhama and Jagdip Singh Sohal Pathobiology of Wild Birds Origin Avian Paramyxovirus Serotype 1
 in Commercial Chickens 
 Muhammad Zubair Shabbir, Sameera Akhtar, Yi Tang, Tahir Yaqub, Arfan Ahmad, Ghulam Mustafa, Muhammad Azhar Alam and Muhammad Munir β-lactamase mediated antimicrobial resistance in Camplobacter isolates: First report of blaOXA-61 gene occurrence from India M. Suman Kumar, Ramees T.P., H. Dhanze, Z.B. Dubal, R.S. Rathore and Ashok Kumar Sero-surveillance of paratuberculosis in domestic ruminant population of western India Mukta Jain, S.V. Singh, K.K. Chaubey, Manju Singh, Saurabh Gupta, G.K. Aseri, Neelam Jain, Parul Yadav, Neeraj Khare, Sujata Jayaraman and J.S. Sohal Evaluation of the comparative performance of three egg based solid media for the primary isolation of Mycobacterium avium subspecies paratuberculosis Mukta Jain, S.V. Singh, G.K. Aseri, Neelam Jain, Parul Yadav, Neeraj Khare, Sujata Jayaraman and J.S. Sohal

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Pre-treatment techniques on selective isolation of Actinobacteria from the coast of Andaman Islands Sumitha Gopalakrishnan, Ranjeet K. , Jai Sunder and S. Venu Immunostimulatory and ameliorative effects of Aloe vera gel extract against infectious bursal disease in white Leghorn chickens G. Elaiyaraja, K. Dhama, M. Asok kumar, M. Palanivelu, Swati Sachan, Deepak Kumar and Yashpal S. Malik Sero-reactivity pattern of secreted proteins of native ‘S 5’ strain to human sera positive for Mycobacterium avium subsp. paratuberculosis infection Saurabh Gupta, Shoor Vir Singh, A.K. Bhatia, Kundan Kumar Chaubey, Manju Singh, Naveen Kumar and Rinko D. Gupta Evaluation of rNS1 based latex agglutination test for sero-diagnosis of Japanese encephalitis in swine M.R. Grace, H. Dhanze, B.R. Gulati, P.B. Pantawane, M. Sivakumar and A. Kumar Do other cellular receptor(s) for edema factor of Bacillus anthracis exists, as evidenced by Protective antigen independent inhibition of embryo growth and angiogenesis Rekha Khandia Amplification of Ferritin-2 by RACE-PCR from Hyalomma anatolicum: a potential vaccine candidate for tick control H.V. Manjunathachar, B.C. Saravanan, Binod Kumar, G. Ravikumar, Madan Mohan and Srikant Ghosh Tick control: identification of cross protective anti-tick vaccine candidates from Hyalomma anatolicum through RNA interference (RNAi) H.V. Manjunathachar, Binod Kumar, B.C. Saravanan and Srikant Ghosh Epidemiological investigation of poultry coccidiosis in and around tarai region of Uttarakhand S. Pant and P. Bhatt Medicinal plants: emerging strategies for gut health, immunity and its development in poultry Jai Sunder, Sujatha Tamilvanan and Anandamoy Kundu Effects of aqueous extract of ornamental plants on diabetic mice Ashok K. Munjal and Nidhi Mishra Feeding Moringa oleifera leaf powder improves semen characteristic and fertility of layer breeders Prabakar G., Gopi M., Kolluri G., Jaydip J.R., Tamilmani T., Beulah Pearlin V. and Jag Mohan Feeding combination of synthetic carotenoids improves heat stress and immune responses in broiler chickens Gopi M., Jaydip J.R., Prabakar G., Gautham K., Shanmathi M. and Tyagi J.S. Tissue distribution and relative gene expression profiles of orphan nuclear receptors related to xenobiotic metabolism in goat and sheep Arun Kumar De, Ramachandran Muthiyan and Dhruba Malakar Clinical management of wedge and complex fracture of long bones in dogs by using composite cancellous bone xenograft Swaroopananda Sahoo, Rekha Pathak, Amarpal, Mudasir Ahmad Shah, Reetu, G. Taru Sharma and Vikash Chandra

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Genetic profiling of Indian poultry rotavirus D isolates, exhibiting circulation of a different lineage Jobin Jose Kattoor, Shubhankar Sircar, Sharad Saurabh, Pallavi Deol, Kuldeep Dhama and Y.P.S. Malik Designing of novel primers based forensic tools for speciation and differentiation of cow and buffalo tissues Hari Singh, Gaya Prasad and Minakshi Prasad Inter- species transmission of bovine coronavirus infection in India Minakshi P., Basanti B., Ikbal and Ranjan K. Predominance occurrence of neglected gastrointestinal viruses in cows in Haryana Basanti B., Minakshi P., Ikbal and Ranjan K. Surveillance of group A rotavirus in small ruminant population of Northern India Shubhankar Sircar, Sharad Saurabh, Jobin J. Kattoor, Pallavi Deol, Kuldeep Dhama and Yashpal S. Malik First time recovery of a novel biotype ‘Indian Bison Type’ of M. paratuberculosis exclusively infecting Indian goat population, from the Mandya breed of sheep suffering from clinical Johne’s Disease Mukartal S.Y., Rathnamma D., Narayanswamy H.D., Singh M., John Stephen B., Gupta S., Kumar Chaubey K., Singh Sohal J., Sahzad and Dhama K. Molecular characterization of bovine viral diarrhea virus (BVDV) infection in bovine fecal samples Minakshi P., Ikbal, Basanti B. and Ranjan K. Molecular characterisation of nodC gene in Promiscous rhizobia Ikbal, Minakshi P., Basanti B., Upender Lambe and V.K. Sikka Development of gold nano particle based immuno dot-blot assay for detection of bluetongue virus Upendera P. Lambe and Minakshi Anti-biogram of Escherichia coli and Salmonella spss isolates from poultry under various farming systems of A&N Islands Sujatha T. and Sunder J. Molecular characterization of bluetongue virus and its putative vectors in India Koushlesh Ranjan, Minakshi Prasad, Basanti Brar, Ikbal and Gaya Prasad First report of bovine coronavirus infection in ovines in India Minakshi P., Basanti B., Ikbal, Ranjan K., Upendara P. Lambe and Manimegalai J. Swinepox outbreak in an organized farm, Uttar Pradesh, India Deenanath Ashokkumar, Arfa Fayaz, Nihar Ranjan Sahoo, Ujjwal Kumar De, Sukdeb Nandi, Rukhsana Bano, R. Mageswary, Ashwini R. Chaple, Sanjeevna Kumari Minhas, Awadh Bihari Pandey and Muthannan Andavar Ramakrishnan Multiple antibiotic resistances among E. coli owing to nontherapeutic use of antibiotics in poultry and livestock farming Vigi Chaudhary Expression of an insecticidal fern protein in cotton protects against whitefly A.K. Shukla, S.K. Upadhyay, M. Mishra, S. Saurabh et al.

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Combating antimicrobial resistance: a drive towards better performance- an overview Hafiz M.N. Iqbal School of Engineering and Science, Tecnologico de Monterrey, Campus Monterrey, Ave. Eugenio Garza Sada 2501, Monterrey, N. L., CP 64849, Mexico

Statement of problem and opportunities The antimicrobial resistance (AMR) is a growing problem in the UK and globally. Developing a range of strategies to reduce reliance on antimicrobials will be a key challenge for the future (www.gov.uk/government/uploads/system/uploads/ attachment_data/file/244058/20130902_UK_5_year_AMR_strategy.pdf). Owing to the antibiotic-resistant, infections now account for 25,000 deaths in Europe alone (European Centre for Disease Prevention and Control), and about 23,000 deaths and over 2 million illnesses in the US (Centers for Disease Control and Prevention), annually. In September 2014, the US President “Obama” signed an Executive Order instructing key health agencies to take action to combat the rise of antibioticresistant bacteria (www.whitehouse.gov/the-press-office/2014/09/18/fact-sheetobama-administration-takes-actions-combat-antibiotic-resistan). In recent years, with an ever-increasing scientific knowledge of infectious diseases caused by various microorganisms, more attention is now being focused towards alternative approaches to control and/or limit such deadly infections. In this context, novel materials with antimicrobial activities are attracting the considerable attention of both academia and industry, especially in the biomedical, and other health-related areas of the modern world (Iqbal et al., 2014; 2015a-d). It has been well-documented in the recent literature that many biological materials are suitable media for growth of microorganisms such as bacteria. According to recent reports, microorganisms could survive on such materials for more than 90 days in a hospital environment. Such a high survival rate of pathogens on the materials having great potential to be used in medical applications may contribute to transmissions of diseases at increased risk (Wang et al., 2014; Zou et al., 2014; Iqbal et al., 2015a-d). Because of the growing consciousness and demands of legislative authorities, the manufacture, to reduce bacterial population in healthcare facilities and possibly to cut pathogenic infections, development of novel anti-microbial active materials which are biocompatible and biodegradable are considered to be a potential solution to such a problematic issue. In this regard, research is underway around the world on the development of ‘greener’ polymer technologies. In recent decades, there has been a growing search for new high-performance products for multipurpose applications in biotechnology at large and biomedical, pharmaceutical and/or cosmeceutical in particular (RuizRuiz et al., 2016). The principle of ‘going green’ has directed this search towards eco-friendly materials with multifunctional features (Iqbal et al., 2015e). Words like renewability, recyclability and sustainability are emphasized in growing scientific

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and ecological awareness. The fact is that legislation authorities are acting as a driving force behind the development of these new type of materials which are antimicrobial and green in nature (Iqbal, 2015). In this perspective, there is an urgent need for the development of green polymeric materials/composites that would not involve the use of the toxic or noxious component in their manufacture and potentially be resistive against the wider community of various microbes to avoid some serious wound contaminations (Iqbal et al., 2015a). One area that has received limited attention so far, but that will gain in importance as naturally conferring antimicrobial agents use becomes further established, is the incorporation of such novel agents into medical materials to provide an antibacterial effect on contact of that material with the target bacterium. Such antimicrobial active biomaterials might have great potential to respond to a new infection before the clinical signs are evident, with the potential to significantly improve patient prognosis. Antimicrobial agents-impregnated materials could be used as medical implants and in applications relevant to hospital hygiene. However, there are also clear industrial and biotechnological requests for materials that are loaded with natural agents that can quickly prevent deleterious microbial action following contamination events. It is intended that a technology platform for future exploitation, e.g. in vivo and ex vivo designs to find out other suitable potential applications such as biomedical implants of these newly developed novel materials, could also be established. AMR resistance issues The increasing challenge to health care attributable to the AMR, therefore, AMR has become a worldwide concern, in recent years (Jindal et al., 2015; Holmes et al., 2016). Broadly speaking, AMR is defined as a temporary and/or permanent capability of a microbial strain and its progeny to resist and/or stay viable and multiply against the medication previously used to treat them. Owing to this notable resistivity and non-susceptibility, microbes have been classified as resistant strains to the concentration of an antimicrobial agent used in practice (Cloete, 2003). Among the potential causes, below are some possible explanations for an increased AMR: 1. The genetic transformation from strain to strain. 2. Biofilm matrix forming potential of several strains. 3. Efflux pumps and other outer membrane structural variations. 4. Enzyme-mediated resistance against, in practice, antimicrobials. 5. Enhanced level of metabolic activity within the biofilm structure. 6. Lower/no perfusion of antimicrobial agents through the biofilm matrix. 7. Adaptability and interaction between antimicrobial agents and biofilm matrix. 8. Excessive/useless consumption of in practice antimicrobials in a random order. 9. Genetic variation and adaptability against the excessive antimicrobials exposure.

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Fig. 1: Illustrates several approaches for combating of antimicrobial resistance (Uchil et al., 2014).

Antimicrobial active materials Bio-based natural materials are moving into the mainstream applications changing the dynamics of 21st-century materials and their utilization in drug delivery strategies. Owing to the increasing consciousness and demands to reduce bacterial contaminations in healthcare facilities and possibly to cut pathogenic infections, the engineering aspects of novel active anti-microbial materials are considered to be a potential solution to such a problematic issue (Iqbal, 2015). These materials have not only been a motivating factor for the materials scientists but also they provide potential opportunities for improving the living standard (Nair and Laurencin, 2007; Iqbal, 2015). In past years, several authors have already been reported antibacterial features of several materials including silver nanoparticles (Michl et al., 2014; Wang et al., 2014; Iqbal et al., 2015b; Lu et al., 2015). However, excess release of silver nanoparticles inhibits osteoblasts growth and can also cause many severe side effects such as cytotoxicity (Wang et al., 2014). Therefore, there is a persistent need to prepare green composites using one or more individual biopolymers to reduce or even eliminate the risk of bacterial infection without impairing the cytotoxicity capabilities. The antibacterial potential of natural phenols, along with their antiseptic characteristics, has already been reported elsewhere (Ultee et al., 2002; Rukmani et al., 2012; Shahidi et al., 2014). Research on several proteins, including collagen, fibroin, keratin, and others is in progress for the development of materials with multifunctional characteristics. Among the natural materials, keratinous proteins are attractive candidates to prepare keratin-based composites which in turn may find potential applications in bio-medical, pharmaceutical, tissue engineering, and cosmetic industries (Khosa and Ullah 2013). By these evidences, we hypothesized that natural phenols are among the practical choice for inhibiting bacterial infections

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and investigated the antibacterial features of these compounds, incorporated materials. Figure 2 illustrates a development and antibacterial behavior of phenol-gkeratin-EC based materials (Iqbal et al., 2015b).

Fig. 2: The design and antibacterial behavior of phenol-g-keratin-EC based materials (Iqbal et al., 2015b).

The antibacterial mechanism of natural phenols is naturally concomitant due to the presence of active hydroxyl groups. This is because the interaction between natural phenols and bacteria can change the metabolic activity of bacteria and eventually cause their death (Iqbal et al., 2015). Based on an earlier published data, most of the phenolic compounds including gallic acid, p-4-hydroxybenzoic acid, and thymol have an ability to disrupt the lipid structure of the bacterial cell wall, further leading to a destruction of the cell membrane, cytoplasmic leakage, and cell lysis which ultimately leads towards the cell death (Veras et al., 2012; Milovanovic et al., 2013; Shahidi et al., 2014). Furthermore, the delocalization of the electrons on their structure has also been reported to contribute to their antibacterial activity as well (Ultee et al., 2002; Elegir et al., 2008). Concluding remarks and future considerations In summary, the present work was aimed to critically overview the literature to establish the stability and infective capacity of the naturally occurring antimicrobial agents impregnated materials using a range of microbiological techniques against Staphylococcus aureus, Escherichia coli, Pseudomonas aeruginosa and other pathogenic bacteria (including antibiotic-resistant forms). The present review work also aimed at combatting AMR, and research that underpins the development of strategies to mitigate the effects e.g. through novel alternatives to antimicrobials. Whereas, the antimicrobials are defined as any compound (natural, synthetic or

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semi-synthetic) with a direct action on microorganisms, used in the treatment or prevention of infections or maintenance of health. Through judicious design, novel characteristics of the developed material can be modified to achieve optimal infective capability and therefore enhanced antibacterial control. Such materials include but not limited to the biodegradable and biocompatible films and highly porous 3-D constructs. Moreover, a novel type of potent materials could be designed for the management and skin regeneration/repair from injury, particularly burns and ulcers, where the risk of bacterial infection is high. Material structure and performance integrity needs to be accessed using a range of analytical and imaging techniques. References Cloete, T.E. (2003). Resistance mechanisms of bacteria to antimicrobial compounds. International Biodeterioration & Biodegradation, 51(4): 277-282. Elegir, G., Kindl, A., Sadocco, P. and Orlandi, M. (2008). Development of antimicrobial cellulose packaging through laccase-mediated grafting of phenolic compounds. Enzyme and Microbial Technology, 43(2): 84-92. Holmes, A.H., Moore, L.S., Sundsfjord, A., Steinbakk, M., Regmi, S., Karkey, A. and Piddock, L.J. (2016). Understanding the mechanisms and drivers of antimicrobial resistance. The Lancet, 387(10014), 176-187. https://www.whitehouse.gov/the-press-office/2014/09/18/fact-sheet-obama-administration-takesactions-combat-antibiotic-resistan [Last accessed: October 04, 2016]. Iqbal H.M.N., Kyazze, G., Locke, I.C., Tron, T. and Keshavarz, T. (2015a). Development of biocomposites with novel characteristics: Evaluation of phenol-induced antibacterial, biocompatible and biodegradable behaviours. Carbohydrate Polymers, 131: 197-207. Iqbal H.M.N., Kyazze, G., Locke, I.C., Tron, T. and Keshavarz, T. (2015c). Development of novel antibacterial active, HaCaT biocompatible and biodegradable CA-gP (3HB)-EC biocomposites with caffeic acid as a functional entity. Express Polymer Letters, 9: 764-772. Iqbal H.M.N., Kyazze, G., Locke, I.C., Tron, T. and Keshavarz, T. (2015d). Poly (3hydroxybutyrate)-ethyl cellulose based bio-composites with novel characteristics for infection free wound healing application. International Journal of Biological Macromolecules, 81, 552-559. Iqbal, H.M.N. (2015). Development of bio-composites with novel characteristics through enzymatic grafting. Doctoral dissertation, University of Westminster, London, UK. Iqbal, H.M.N., Kyazze, G., Locke, I. C., Tron, T. and Keshavarz, T. (2015b). In situ development of self-defensive antibacterial biomaterials: phenol-g-keratin-EC based bio-composites with characteristics for biomedical applications. Green Chemistry, 17(7), 3858-3869. Iqbal, H.M.N., Kyazze, G., Tron, T. and Keshavarz, T. (2014). “One-pot” synthesis and characterisation of novel P (3HB)-ethyl cellulose based graft composites through lipase catalysed esterification. Polymer Chemistry,5(24), 7004-7012. Iqbal, H.M.N., Kyazze, G., Tron, T. and Keshavarz, T. (2015e). Laccase‐assisted approach to graft multifunctional materials of interest: Keratin‐EC based novel composites and their characterisation. Macromolecular Materials and Engineering, 300(7), 712-720. Jindal, A.K., Pandya, K. and Khan, I.D. (2015). Antimicrobial resistance: A public health challenge. Medical Journal Armed Forces India, 71(2), 178-181. Khosa, M. A. and Ullah, A. (2013). A sustainable role of keratin biopolymer in green chemistry: a review. J Food Processing and Beverages, 1(1), 8.

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Lu, Z., Zhang, X., Li, Z., Wu, Z., Song, J. and Li, C. (2015). Composite copolymer hybrid silver nanoparticles: preparation and characterization of antibacterial activity and cytotoxicity. Polymer Chemistry, 6(5): 772-779. Michl, T.D., Locock, K.E., Stevens, N.E., Hayball, J.D., Vasilev, K., Postma, A. and Griesser, H.J. (2014). RAFT-derived antimicrobial polymethacrylates: elucidating the impact of endgroups on activity and cytotoxicity. Polymer Chemistry, 5(19): 5813-5822. Milovanovic, S., Stamenic, M., Markovic, D., Radetic, M. and Zizovic, I. (2013). Solubility of thymol in supercritical carbon dioxide and its impregnation on cotton gauze. The Journal of Supercritical Fluids, 84: 173-181. Nair, L.S., and Laurencin, C.T. (2007). Biodegradable polymers as biomaterials. Progress in Polymer Science, 32(8), 762-798. Ruiz-Ruiz, F., Mancera-Andrade, E.I. and Iqbal H.M.N., (2016). Marine-Derived Bioactive Peptides for Biomedical Sectors-A Review. Protein & Peptide Letters. In Press. http://www.ncbi.nlm.nih.gov/pubmed/27491381. Rukmani, A. and Sundrarajan, M. (2012). Inclusion of antibacterial agent thymol on βcyclodextrin-grafted organic cotton. Journal of Industrial Textiles, 42(2), 132-144. Shahidi, S., Aslan, N., Ghoranneviss, M. and Korachi, M. (2014). Effect of thymol on the antibacterial efficiency of plasma-treated cotton fabric.Cellulose, 21(3), 1933-1943. Uchil, R.R., Kohli, G.S., KateKhaye, V.M. and Swami, O.C. (2014). Strategies to combat antimicrobial resistance. Journal of Clinical and Diagnostic Research: JCDR, 8(7), ME01. UK Five Year Antimicrobial Resistance Strategy 2013 to 2018 (PDF). https://www.gov.uk/government/uploads/system/uploads/attachment_data/file/244058/20130902_ UK_5_year_AMR_strategy.pdf [Last accessed: October 04, 2016]. Ultee, A., Bennik, M.H.J. and Moezelaar, R. (2002). The phenolic hydroxyl group of carvacrol is essential for action against the food-borne pathogen Bacillus cereus. Applied and environmental Microbiology, 68(4), 1561-1568. Veras, H.N., Rodrigues, F.F., Colares, A.V., Menezes, I.R., Coutinho, H.D., Botelho, M.A. and Costa, J.G. (2012). Synergistic antibiotic activity of volatile compounds from the essential oil of Lippia sidoides and thymol. Fitoterapia, 83(3): 508-512. Wang, L., He, S., Wu, X., Liang, S., Mu, Z., Wei, J. and Wei, S. (2014). Polyetheretherketone/ nano-fluorohydroxyapatite composite with antimicrobial activity and osseointegration properties. Biomaterials, 35(25): 6758-6775. Zou, K., Liu, Q., Chen, J. and Du, J. (2014). Silver-decorated biodegradable polymer vesicles with excellent antibacterial efficacy. Polymer Chemistry, 5(2): 405-411.

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Fresh water blues: “brain-eating amoeba” in Indian subcontinent Sunil K. Joshi Frank Reidy Research Center of Bioelectrics College of Health Sciences, Old Dominion University 4211 Monarch Way, IRP-2, Suite # 300 Norfolk, VA 23508 USA

Introduction Indian subcontinent (India, Pakistan, Bangladesh, Nepal, Bhutan and Sri Lanka) mostly has tropical climate and global warming has added more days to summer. Such warm climate is perfect for amoeba to become a deadly infection with 98% mortality. This almost universally fatal disease known as ‘Primary Amebic Meningoencephalitis (PAM)’ or ‘Amebic Encephalitis’ which is caused by a thermophile ameboflagellate, Naegleria fowleri aka ‘Brain-eating amoeba’(1-3). Due to low incidence rate, the dangers of this reclusive infection are underestimated and ignored. In past few years, number of cases worldwide has increased significantly due to global climate change, particularly ‘El Nino’ effect is playing major role. The Naegleria fowleri is climate-sensitive which enters through nose either during swimming or performing ‘neti’ (nasal rinse) and ablution, a ritual cleansing that includes nasal passages(2). This has been now confirmed that city water supply through household faucets also contain Naegleria fowleri and pose more significant health concerns. In cold water, this ameboflagellate remains in dormant form as a cyst which transform into infective flagellate form when water becomes warm. Once enter through nasal mucosa, the Naegleria fowleri migrates to the brain via the olfactory nerve and cause lethal neuro-inflammation and damage to the brain. Whether Naegleria can enter through the open wounds is not known. However, one cannot get infected with Naegleria from swallowing contaminated water(3). Historically, the very first existence of brain-eating ameba was reported in Ireland, in 1909. Later, in 1965, two physicians named, R.F. Carter and M. Fowler from Australia, formally studied and identified the Naegleria fowleri as a causative agent of PAM(1). In 1978, CDC created the national Free-living Ameba (FLA) Laboratory, which has become a national resource and global leader for providing diagnostic and clinical guidance as disease is rare. The CDC began formally tracking Naegleria fowleri infections since 1989(2). After entering through nose, Naegleria migrates along the olfactory nerve via disruption of the olfactory mucosa and penetration through the submucosal nervous plexus(4). Finally organisms passage through the cribriform plate to frontal lobes of the brain. Once in the brain, the Naegleria destroy brain tissue and cause severe neuro-inflammation in the form of PAM(4-5). Scientists suggest that once in brain, Naegleria organisms produce two proteases enzymes and toxins that dissolve proteins leading to brain hemorrhage and severe tissue necrosis. New research reveals that the brain damage is substantially caused by hyper immune response from the host rather by Naegleria itself(4-5). This amplified immune response is

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mainly due to prior exposure to the antigens and larger size of these pathogens. This overwhelmed immune response, which is dominated by acute inflammatory cytokines, often causes the leakiness of the blood brain barrier (BBB) and severe damage to neuronal tissues(6). The further irreversible brain damage is done by toxins and enzymes secreted by the pathogen(4-6). The PAM is a fulminant central nervous system infection and incubation period can range from 2-15 days(4). Initially infection start with mild symptoms such as headache, fever, nausea, or vomiting, which later becomes complicated very quickly. Death typically occurs within 5 days of symptom onset. The pathogenesis of PAM is quite similar to bacterial meningitis and possibly misdiagnosed most of the time and underreported. As in the case of bacterial meningitis, fulminant PAM manifests as high fever and severe headaches, nausea, vomiting, stiff neck, confusion, lack of attention to people and surroundings, loss of balance followed by a rapid progression to seizures, hallucinations or coma. Since the incidence of PAM is rare, it is difficult to diagnose and >75% of diagnoses are usually confirmed after the death during autopsy. Diagnosis of PAM usually start by taking detailed history and observing cardinal signs of meningitis. Early symptomatic diagnosis of meningitis is performed by patient’s physical evaluation and observing Kernig and Brudzinski signs(5-7). The Kernig’s signs represent the resistance or pain during extension of the patient’s knees beyond 135 degrees due to spasm of the hamstring muscles. While a positive Brudzinski’s sign represent if passive flexion of the neck induce reflex flexion of the patient’s hips and knees(4-6). Currently there is no rapid diagnostic test is available and it take longer than a week to identify. In the United States, only few laboratories can diagnose the disease using specific laboratory tests. These special labs are directed through national FLA lab of CDC. The diagnosis of PAM and identification of Naegleria can be performed by direct visualization of motile organisms in cerebrospinal fluid (CSF), biopsy, or tissue specimens. However, the available new tests can identify specific antigens (by indirect immunofluorescence/immunohistochemistry) and nucleic acid (by PCR) of Naegleria extracted from CSF or tissue specimens(7-8). The ‘brain-eating ameba’ related sickness is rare and often results into sudden and tragic death in almost every case. Since pathogenesis of Naegleria is relatively under studied, no specific treatment for PAM is available yet and line of treatment mainly limited to treating symptoms and apply empirical therapy(7-8). Very few cases have been treated and survived. If medical help is available at early stage, there is possibility to treat PAM by available anti-parasitic drugs alone or in combination with antibiotics(7-8). The drugs of choice must be able to effectively cross the bloodbrain barrier to target parasites residing deep in the brain tissue. Studies have shown the effectiveness of various drugs such as azole antifungals (e.g., Ketoconazole and Itraconazole); Diamidines (Pentamidine); Cotrimoxazole (a combination of two antibacterial medicines- a sulfonamide medicine called sulfamethoxazole, and trimethoprim). Also, the antifungal agent Amphotericin B is the drug of choice for PAM and is administered intravenously and intrathecally, usually in combination with anti-tuberculosis drug Rifampicin(9) and antineoplastic/ anti-leishmaniasis agent 8 

 

Miltefosine (alkylphosphocholine compound). Due to the severe side effects of these drugs, there is an urgency to develop new, cost-effective and safe drugs that can cross the blood-brain barrier. Modern computational tools and omics approaches may be used in high-throughput phenotypic screening, specifically for pathogenic N. fowleri(10-11). Founding father and skilled scientist Benjamin Franklin shared the axiom, “An ounce of prevention is worth a pound of cure” implies in many situations in life including dealing with ‘Summertime Sadness’ incidences. The heartbreaking and devastating events due to exposure of Naegleria, particularly in young adults can be prevented very effectively by following the simple common sense precautions. References 1.

Fowler M and Carter RF. (1965). Acute pyogenic meningitis probably due to Acanthamoeba sp.: a preliminary report. Br Med J 2:740-742. 2. Yoder JS, Straif-Bourgeois S, Roy SL, Moore TA, Visvesvara GS, Ratard RC, et al. (2012). Primary amebic meningoencephalitis deaths associated with sinus irrigation using contaminated tap water. Clin Infect Dis 55: e79-85. 3. Siddiqui R, Khan NA. (2014). Primary amoebic meningoencephalitis caused by Naegleria fowleri: an old enemy presenting new challenges. PLoS Negl Trop Dis. 14;8(8):e3017. 4. Wiwanitkit V. (2004). Review of clinical presentations in Thai patients with primary amoebic meningoencephalitis. MedGenMed. 8;6(1):2. 5. Cervantes-Sandoval I, Serrano-Luna Jde J, García-Latorre E, Tsutsumi V and Shibayama M. (2008). Characterization of brain inflammation during primary amoebic meningoencephalitis. Parasitol Int. 57(3):307-13. 6. Baig AM. (2015). Pathogenesis of amoebic encephalitis: Are the amoebae being credited to an ‘inside job’ done by the host immune response? Acta Trop. 148:72-6. 7. Capewell LG, Harris AM, Yoder JS, Cope JR, Eddy BA, Roy SL, Visvesvara GS, Fox LM and Beach MJ. (2015). Diagnosis, Clinical Course, and Treatment of Primary Amoebic Meningoencephalitis in the United States, 1937-2013. J Pediatric Infect Dis Soc. 4(4):e68-75. 8. Grace E, Asbill S and Virga K. (2015). Naegleria fowleri: pathogenesis, diagnosis, and treatment options. Antimicrob Agents Chemother. 59(11):6677-81. 9. Sood A, Chauhan S, Chandel L and Jaryal SC. (2014). Prompt diagnosis and extraordinary survival from Naegleria fowleri meningitis: A rare case report. Indian J Med Microbiol. 32:193-6 10. Vargas-Zepeda J, Gómez-Alcalá AV, Vásquez-Morales JA, Licea-Amaya L, De Jonckheere JF and Lares-Villa F. (2005). Successful treatment of Naegleria fowleri meningoencephalitis by using intravenous amphotericin B, fluconazole and rifampicin. Arch Med Res. 36(1):83-6. 11. Baig AM and Khan NA. (2014). Novel chemotherapeutic strategies in the management of primary amoebic meningoencephalitis due to Naegleria fowleri. CNS Neurosci Ther. 20(3): 289-90.

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Genetics and vaccine efficacy of wild birds origin avian paramyxoviruses in domestic chickens Muhammad Zubair Shabbir1, Sameera Akhtar1, Yi Tang2,Tahir Yaqub1, Arfan Ahmad1, Ghulam Mustafa1, Muhammad Azhar Alam1 and Muhammad Munir3 1

University of Veterinary and Animal Sciences Lahore 54600, Pakistan; The Pennsylvania State University, University Park, PA 16802, USA; 3 The Pirbright Institute, Woking, Surrey, GU24 0NF, United Kingdom 2

Introduction Newcastle disease (ND) is one of the most important and highly contagious viral diseases of domestic poultry and wild birds (Alexander, 1998), and is caused by the ND virus (NDV). NDV, thetype-species of avian paramyxovirus serotype 1 (APMV-1), is an enveloped, negative-sense, non-segmented, single-stranded RNA virus of the genus Avulavirus within the family Paramyxoviridae (Alexander, 1998; Kolakofsky et al., 2005). The genome of all APMVs is approximately 15.2 kilobases in length and encodes the nucleoprotein (N), phosphoprotein (P), matrix protein (M), fusion protein (F), haemagglutinin-neuraminidase (HN), and large polymerase (L) genes, in addition to the V protein that is expressed by the RNA editing process of the P gene (Kolakofsky et al., 2005). Based on the analysis of F gene sequences, all APMV-1strains can be divided into either lineages or genotypes (Aldous et al., 2003; Cattoli et al., 2010; Czegledi et al., 2006; Diel et al., 2012; Kim et al., 2007; Munir et al., 2012b; Perozo et al., 2008; Snoeck et al., 2009). Currently, we have also characterized a velogenic strain of NDV from clinically asymptomatic birds, adding further complexity to the pathobiology of NDVs in different avian species (Munir et al., 2012b). These studies, mainly focused on virus genetics, prompted us to determine and evaluate the transmissibility, clinical impact and to assess the potential threats of APMVs isolated from wild birds, in domestic chickens. From the data presented here, we conclude that the APMV-1 and PPMV-1 viruses isolated from wild birds are fully infectious and pathogenic for the domesticated chicken, and that these strains can potentially be transmitted between infected and healthy birds. Moreover, commonly applied vaccines are unable to fully protect the vaccinated birds from the clinical disease induced by wild bird-origin APMV-1 and PPMV-1, since virus shedding was not contained despite the evidence of seroconversion. Based on the viral bioinformatics analysis, we highlight the circulation of diverse genetic clusters of APMV-1 and PPMV-1 in Pakistan. Materials and Methods Sampling History and Virus Stocks Clinical samples from captive wild birds were collected from two independent outbreaks in private farms around the Lahore district, Punjab, Pakistan. Trachea, lungs, spleen and caecal tonsils from dead birds (n=15) of each flock were collected

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aseptically, pooled and processed for virus isolation through the chorioallantoic sac route of embryonated eggs (five times for pigeon and four times for peacock isolates) (Alexander and Swayne, 1998) followed by identification through spot haemagglutination assay and polymerase chain reaction, as we described earlier (Munir et al., 2012b; Shabbir et al., 2013b). Next Generation Sequencing (NGS) From each isolate, viral RNA was extracted separately using a commercially available RNA extraction kit (QIAamp Viral RNA Mini Kit, Qiagen, USA) as per the manufacturer’s instructions. Quantity (NanoDrop, USA) and quality (QubitFlourometer, USA) of extracted RNA were measured and subjected to NGS based whole genome sequencing (Macrogen Inc., Seoul, Korea). Briefly, a library was prepared with 1µg of total RNA by IlluminaTruSeq mRNA Sample Prep kit (Illumina, Inc., San Diego, CA, USA). The RNA was fragmented using divalent cations under elevated temperature. Using SuperScript II reverse transcriptase (Invitrogen, USA) and random primers, the cleaved RNA fragments were copied into first and second strand cDNA synthesis using DNA polymerase I and RNase H. The cDNA fragments were then subjected to an end-repair process, the addition of a single ‘A’ base, and ligation of the indexing adapters. The products were purified and enriched through PCR to create a final cDNA library. The libraries were quantified using qPCR quantification protocol guide (KAPA Library Quantification kits for Illumina Sequencing platforms) and qualified using the TapeStation D1000 ScreenTape (Agilent Technologies, Waldbronn, Germany). Indexed libraries were then sequenced through a HiSeq2500 platform (Illumina, San Diego, USA). Adaptors were trimmed from the raw data and reads mapping to contaminants (rRNA, chicken or human sequences) were removed using both sort MeRNA (Kopylova et al., 2012) and BWA-MEM methods (Li & Durbin, 2010). Unmatched sequence reads were assembled using de novo SPAdes assembly software (version 3.5.0) (Bankevich et al., 2012). All assembled contiguous sequences (contigs) were aligned to the reference genome (LaSota strain, accession number AY845400) using LASTZ (Harris, 2007a; Harris, 2007b) to identify and extract maximally aligned viral contigs. To further improve contigs, all raw reads of each segment were mapped back to the assembled contigs. Finally, the consensus sequences from the remapping reads and LASTZ contig alignment were obtained using SAMtools command lines (Li et al., 2009). These sequences were submitted to GenBank and are available under accession numbers KU885948 (Peacock/MZS-UVAS-Pak/2014) and KU885949 (Pigeon/MZS-UVAS-Pak/2014). Sequence and Phylogenetic Analysis Complete genome sequences of reference strains of each recognized lineage (Aldous et al., 2003) and genotype (Diel et al., 2012) of APMVs as well as the vaccine strain were used to determine percentage similarity of coding regions together with detailed analysis of deduced residues for F and HN protein. To elucidate the phylogenetic relationships between APMV-1 and PPMV-1 viruses

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reported here and characterized previously from Asia and other parts of the world, we first compiled a dataset of available complete genomes of APMV-1 and PPMV1. For global and high-level clustering patterns, we next collected the 3’ hypervariable region (374bp) of F gene from all available sequences. Both these datasets were aligned in BioEdit version 5.0.6 (Hall, 1999) using ClustalW and were edited to equal lengths. All sequences that aligned poorly or with incomplete information were excluded from the analysis. The phylogenetic relationships of Peacock/MZS-UVAS-Pak/2014 and Pigeon/MZS-UVAS-Pak/2014 with APMVs reported previously around the globe were determined by constructing Bayesian Inference with the program MrBayes version 3.1.2 (Ronquist & Huelsenbeck, 2003). Two independent Monte Carlo Markov (MCM) chains were executed and sampled every 1000 generations using the default parameters of the priors’ panel. The analysis was based on the GTR + I + G model, which allow significantly changed posterior probability estimates. Phylogenetic relationship was also established with the MEGA version 6.0 software programme using the neighbor-joining method with the Kimura 2-parameter model. The evolutionary distances were inferred using the pairwise distance method and expressed as the number of nucleotide substitutions per site giving a statistical significance of the tree topology by 1000 bootstrap resampling of the data (Tamura et al., 2013). For clarity on association of these viruses with previously characterized Pakistani strains, the lineage-based nomenclature was used in this study as described by Aldous et al. (2003). The corresponding genotypes were also displayed where needed for comparison purposes (Aldous et al., 2003). Immunization and Challenge Experiment Clinicopathological assessments of the isolates in immunized, challenged, mock-infected and contact birds were performed individually. Eighty one-day-old chicks (Hubbard) were procured from commercial hatchery and raised until the end of the experiment (day 40) at the Experimental Unit in the Department of Microbiology, University of Veterinary and Animal Sciences, Lahore, Pakistan. Feed and water were provided ad libitum along with general animal care by the dedicated service staff. Presence of maternal antibodies was assessed in all birds on day 1 by haemagglutination inhibition (HI) assay (Alexander, 1998). All birds were divided into two groups as vaccinates and non-vaccinates (n = 40 each) and housed separately. For each isolate, birds in non-vaccinates were divided into three groups named challenged (Ch, n = 10), contact (Contact, n = 5) and mock-infected (Mock, n=5). Birds in vaccinates (VCh, n=20) were kept in groups of 20 for each isolate. Replicating the vaccination schedule commonly practiced by the broiler industry in the country, the birds in VCh group were administered LaSota vaccine (Laprovet, France) twice on day 7 and 25 and one intramuscular injection of killed vaccine (genotype VII) on day 11 as per the manufacturer’s instructions. The lypholized vaccine (LaSota, 1000 doses), used for primary immunization, was dissolved in vaccine-provided sterile buffer and administered via eye drops individually to each bird. For boosting, a nationally

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manufactured and widely used in the field oil-based killed vaccine was applied. The vaccine was administered with a dose of 0.3mL/bird via subcutaneous route around the neck region. Determination of Antibodies Titers Sera samples obtained from birds before and after the challenge were tested for antibodies against NDV using haemagglutination inhibition assay described by the OIE Manual of Standard Diagnostic Tests (Afonso, 2012). Titers were calculated as the highest reciprocal serum dilution giving complete inhibition in 96-well microtiter plate and antibody titers (1:8 or 23) or lower were considered negative for NDV/APMVs (Miller et al., 2013). At the end of the experiment, on day 40, all birds in VCh group were sacrificed and blood was processed for HI assay. Evaluation of Virus Shedding and Viscerotropism The virus shedding from contact birds and tissue tropism in VCh and Ch groups was determined through previously described assays for identification of velogenic and mesogenic strains of NDVs (Wise et al., 2004). Virus shedding was evaluated through oropharyngeal and cloacal swabs, whereas various organs/tissues such as whole blood, brain, Harderian gland, tongue, trachea, lung, heart muscle, breast muscle, hair follicles, gizzard, proventriculus, liver, kidney, spleen, bone marrow, intestine, caecal tonsils, bursa and cloacal tissues were processed to track the distribution of challenged viruses. Histopathology Selected tissues were collected and fixed by immersion in 10% neutral buffered formalin at room temperature for 48 hours followed by processing and embedding in paraffin wax. Tissues section of 5µm were stained with Haematoxylin & Eosin and examined for microscopic lesions under light microscope. Results Wild Birds-Origin APMV-1Strainsare Genetically Divergent Compared to Vaccine Strains The consensus complete genome of both pigeon and peacock-origin APMV-1 were submitted to GenBank under the accession number KU885948 (Peacock/MZSUVAS-Pak/2014) and KU885949 (Pigeon/MZS-UVAS-Pak/2014). The genome lengths of pigeon and peacock isolates followed the ‘rule-of-six’, the characteristic essential feature for efficient replication of paramyxo viruses (Kolakofsky et al., 2005).As expected for AMPV-1 and PPMV-1, the characterized strains showed the order of six open reading frames; 3΄-N-P-M-F-HN-L-5΄. The nucleotide identity between the studied isolates was 86.6%, and these isolates shared a varying degree of genetic diversity with representative genotypes/lineages of APMVs. Comparison to full-length deduced amino acid sequence of F protein of pigeon and peacock isolate showed a divergence of 11.6

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and 11.8 to genotype II (LaSota) and 9.8 and 9.3 to genotype III (Muketeswar), respectively. Pigeon and Peacock Isolates Belong to Genotype VI and Genotype VII To determine epidemiological clustering of isolates in the current study with the APMV-1 and PPMV-1 reported in the public domain, all available complete genomes were downloaded from NCBI databases and used for the phylogenetic analysis and comparative genomics. APMV-1 isolated from the peacock shared maximum genetic similarity with an isolate originating from lineage 5 (genotype VII) while the isolate from the pigeon shared genetic resemblance to isolates originating from lineage 4 (genotype VI). Since most of the reported strains of APMVs and PPMVs are characterized based on the 3 hypervariable region of F gene (374bp), we next analyzed the entire datasets of F gene (~3000) sequences. The Bayesian consensus phylogenetic analysis, verified by the neighbor-joining method, clearly divided the APMV-1 strains into 6 lineages, and Peacock/MZS-UVASPak/2014 isolate clustered with isolates oflineage 5 in association with strains reported previously from Pakistan whereas the Pigeon/MZS-UVAS-Pak/2014 isolate resembled to lineage 4 along with isolates reported from Russia. This isolate shared highest genetic identity with the NDV/Altai/pigeon/770/2011, recently reported from Russian pigeons (Yurchenko et al., 2015). The clustering pattern at a higherresolution was performed with selected isolates within lineage 4/genotype VI and lineage 5/genotype VII showing their close association within their sub-genotypes/ sub-lineages. Based on sequence comparison, both isolates shown more than 15% nucleotide divergence compared to routinely used vaccines in the country; [LaSota (genotype II) and Muketeswar (genotype III)]. Taken together, distinct grouping and level of genetic divergence of these isolates are in agreement with our studies on previously characterized isolates from wildbirds, commercial and backyard poultry in Pakistan. These differences warrant future studies to evaluate the contribution of genetic differences on the protective efficacy of vaccines. Routinely used Attenuated and Killed Vaccines Provide Partial Protection against Field Stains of APMV-1 The protective efficacy of commonly used vaccine type (LaSota and indigenously produced killed vaccines) and routinely used vaccine schedule were assessed individually against pigeon and peacock-originated APMVs. The birds used in the experiment had a maternal antibody geometric mean titer (GMT) of 4.2 before the oral administration of LaSota on day 7. When examined on day 27 before challenge, vaccinated pigeon and peacock groups (VCh) showed a mean titer of 7.15 and 7.40, respectively, indicating potency as well as appropriate application of vaccines to the birds. We observed variations in severity and duration of clinical signs between challenge isolates and over time post infection. Briefly, commonly observed signs were general sickness (depression, anorexia), mild respiratory sounds, oculonasal discharge and reluctance to move. Although severity of clinical signs was more often

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observed in birds challenged with the virulent peacock-derived viruses, the disease outcome coupled with the gross pathognomonic lesions were comparable for both isolates. The group without pre-vaccination (Ch) showed severe clinical picture with all birds dead or killed by 7 days post-infection (dpi) for both isolates. Sudden death with no-to-mild clinical signs was observed particularly in Ch groups and one in VCh group challenged with the peacock isolate. Both the isolates induced nervous signs that included neurological signs such as twisting of the head and neck together on one side, circling movement and opisthotonus. The clinical signs started to appear on 6 dpi in group that was challenged (Ch) with the peacock isolate and by 7dpi in birds challenged with the pigeon isolate. Interestingly, the situation was reversed for VCh group; nervous signs were observed on day 9 dpi in birds challenged with pigeon isolate compared to the group challenged with peacock isolate on day 10 dpi. The Ch group showed mild clinical signs till end of the experiment trial; however the characteristic necropsy lesions were observed on day 8 and day 10 dpi in birdskept with Ch group of peacock and pigeon isolates, respectively. Both strains caused detectable histopathological lesions in affected birds. The microscopic changes were relatively less pronounced in birds challenged with pigeon isolate than peacock isolate. We found hemorrhages in some areas of tracheal tissue coupled with degenerative changes in the lamina propria and absence of pseudo-stratified columnar epithelium. Severe congestion particularly in parabronchial blood capillaries was observed in lung tissues. Liver showed fatty changes evidenced by vacuoles of varying sizes in the cytoplasm of many hepatocytes. The portal vein including the sinusoidal capillaries was engorged with red blood cells and, in some areas, Kupffer’s cells were also observed. Congestion, hemorrhages, degeneration and loss in lymphoid follicles coupled with sloughing of columnar epithelium were evident in caecal tonsils. The microscopic examinations of kidney revealed sever congestion in peritubular capillaries, cellular swelling in renal epithelial cells and coagulative necrosis in renal tubules. Some renal tubules had epithelial cells separated from the basement membrane. Intestine revealed sloughing of epithelial cells in the lumen; a vast majority of the intestinal villi were necrotic and degenerated. Discussion The genotypes VI and VII represent the most prevalent group of APMVs in South East Asia and all APMVs isolated from wild captive birds either belongs to genotype VI or VII (Kim et al., 2007). Two APMVs isolate recovered from pigeon and peacock flock were classified as genotype VI/lineage 4 and VII/lineage 5 compared to the vaccine strains (genotype II/lineage 1 and 2). Analysis of the deduced residues at the cleavage site for the isolates in the current study and previously reported isolates from Pakistan indicated co-circulation of different genotypes of APMVs in the country with similar pathogenicity (based on the motif in the cleavage site). This is of importance since invariability in residue pattern has been observed for velogenic NDVs in a given geographical location (Samuel et al., 2013).

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Although severity of observed clinical signs was relatively less for the pigeon isolate than the peacock isolate, the morbidity, mortality and virus shedding was comparable between these groups. Interestingly, all birds in the non-vaccinated group challenged with peacock isolate died on day 7 dpi due to severe neurological disease. The nervous signs were also prominent in the vaccinated group, potentially indicating lack of complete protection induced by the vaccine. While comparing pre- and post-challenge antibody titers, we found varying but an increasing immune response indicating that birds were sufficiently exposed to the virus antigen. The immune response generated by challenge with the pigeon isolate was greater than that generated by the peacock isolate indicating its efficient replication. Further nervous signs were evident one day earlier in birds challenged with the pigeon isolate compared to the peacock isolate. A potential reason could be the fact that both group of vaccinates were administered killed vaccine containing genotype VII that may have hindered replication of challenged virus of genotype VII more than genotype VI. Viral shedding together with increase in antibody titer suggests that the commonly practiced vaccine schedule and vaccine types (LaSota and killed vaccine of genotype VII) give partial protection from disease, and are unable to protect from infection and virus replication, at least in experimental conditions. Moreover, a lower cross-reactivity was observed between sera collected from LaSota-immunized birds and isolates collected from pigeon and peacock. Taken together, genetic differences observed in the functional domains and neutralization epitopes of the F and HN protein of field isolates and vaccine strains could be attributed to increased virulence and escape from vaccines. Conclusions Two isolates, originated from pigeon and peacock, were genotypically and pathobiologically characterized. Both isolates had the potential to cause disease and subsequent virus shedding even in vaccinated birds. The results presented may be useful in revising the vaccine schedule being practiced currently in Pakistan and other NDV-endemic countries. Furthermore, it ascertains the need to establish and maintain active surveillance for NDVs in wild birds. References Afonso, C. L., Miller, P.J., Grund, C., Koch, G., Peeters, B., Selleck, P.W., Srinivas, G.B. (2012). OIE Manual of Diagnostic Tests and Vaccines for Terrestrial Animals, 7th edn, pp. 555-573. Edited by OIE. Paris. Aldous, E.W., Fuller, C.M., Mynn, J.K. and Alexander, D.J. (2004). A molecular epidemiological investigation of isolates of the variant avian paramyxovirus type 1 virus (PPMV-1) responsible for the 1978 to present panzootic in pigeons. Avian Pathol 33, 258-269. Aldous, E.W., Mynn, J.K., Banks, J. and Alexander, D.J. (2003). A molecular epidemiological study of avian paramyxovirus type 1 (Newcastle disease virus) isolates by phylogenetic analysis of a partial nucleotide sequence of the fusion protein gene. Avian Pathol 32, 239256. Alexander, D. J. (2013). Newcastle disease, other avian paramyxoviruses, and pneumovirus infections. In Diseases of Poultry, 4th edn, pp. 63-99. Edited by B. Saif. Y. M., H. J., Glisson, J. R., Fadly, L. R. M., Swayne, D. Ames: Iowa State University Press.

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Alexander, D. J., Purchase, H. G., Arp, L. H., Domermuth, C. H., Pearson, J. E. (1998). Newcastle Disease and other Avian Paramyxovirus. Kennett Square, PA: American Association of Avian Pathologists. Awu, A., Shao, M. Y., Liu, M. M., Hu, Y. X., Qin, Z. M., Tian, F. L. and Zhang, G. Z. (2015). Characterization of two pigeon paramyxovirus type 1 isolates in China. Avian Pathol 44, 204-211. Bankevich, A., Nurk, S., Antipov, D., Gurevich, A. A., Dvorkin, M., Kulikov, A. S., Lesin, V. M., Nikolenko, S. I., Pham, S., Prjibelski, A. D., Pyshkin, A. V., Sirotkin, A. V., Vyahhi, N., Tesler, G., Alekseyev, M. A. and Pevzner, P. A. (2012). SPAdes: a new genome assembly algorithm and its applications to single-cell sequencing. J Comput Biol 19, 455-477. Cattoli, G., Fusaro, A., Monne, I., Molia, S., Le Menach, A., Maregeya, B., Nchare, A., Bangana, I., Maina, A.G., Koffi, J.N., Thiam, H., Bezeid, O.E., Salviato, A., Nisi, R., Terregino, C. and Capua, I. (2010). Emergence of a new genetic lineage of Newcastle disease virus in West and Central Africa-implications for diagnosis and control. Vet Microbiol 142, 168-176. Collins, M. S., Strong, I. and Alexander, D. J. (1994). Evaluation of the molecular basis of pathogenicity of the variant Newcastle disease viruses termed “pigeon PMV-1 viruses”. Arch Virol 134, 403-411. Czegledi, A., Ujvari, D., Somogyi, E., Wehmann, E., Werner, O. and Lomniczi, B. (2006). Third genome size category of avian paramyxovirus serotype 1 (Newcastle disease virus) and evolutionary implications. Virus Res 120, 36-48. Diel, D.G., da Silva, L.H., Liu, H., Wang, Z., Miller, P.J. and Afonso, C.L. (2012). Genetic diversity of avian paramyxovirus type 1: proposal for a unified nomenclature and classification system of Newcastle disease virus genotypes. Infect Genet Evol 12, 1770-79. Dortmans, J. C., Koch, G., Rottier, P. J. and Peeters, B. P. (2011). A comparative infection study of pigeon and avian paramyxovirus type 1 viruses in pigeons: evaluation of clinical signs, virus shedding and seroconversion. Avian Pathol 40, 125-130. Dortmans, J. C., Peeters, B. P. and Koch, G. (2012). Newcastle disease virus outbreaks: vaccine mismatch or inadequate application? Vet Microbiol 160, 17-22. Hall, T. A. (1999). BioEdit: a user-friendly biological sequence alignment editor and analysis program for Windows 95/98/NT. Nucleic Acids Symposium Series41, 95-98. Harris, R. S. (2007a). Improved pairwise alignment of genomic DNA. Harris, R. S. (2007b).Improved pairwise alignment of genomic DNA: ProQuest. Hu, S. L., Ma, H. L., Wu, Y. T., Liu, W. B., Wang, X. Q., Liu, Y. L. and Liu, X. F. (2009). A vaccine candidate of attenuated genotype VII Newcastle disease virus generated by reverse genetics. Vaccine 27, 904-910. Hu, S.L., Wang, T.Y., Liu, Y.L., Meng, C., Wang, X.Q., Wu, Y.T. and Liu, X.F. (2010). Identification of a variable epitope on the Newcastle disease virus hemagglutininneuraminidase protein. Vet Microbiol 140, 92-97. Hu, Z. a. X., L. (2015). NDV Induced Immune-Pathology in Chickens. Brit J Virol2, 25-27. Jindal, N., Chander, Y., Chockalingam, A. K., de Abin, M., Redig, P. T. and Goyal, S. M. (2009). Phylogenetic analysis of Newcastle disease viruses isolated from waterfowl in the Upper Midwest Region of the United States. Virol J6. Khan, T. A., Rue, C. A., Rehmani, S. F., Ahmed, A., Wasilenko, J. L., Miller, P. J. and Afonso, C. L. (2010). Phylogenetic and Biological Characterization of Newcastle Disease Virus Isolates from Pakistan. J Clin Microbiol 48, 1892-1894. Kim, L.M., King, D.J., Curry, P.E., Suarez, D.L., Swayne, D.E., Stallknecht, D.E., Slemons, R.D., Pedersen, J.C., Senne, D.A., Winker, K. and Afonso, C.L. (2007). Phylogenetic diversity among low-virulence Newcastle disease viruses from waterfowl and shorebirds and comparison of genotype distributions to those of poultry-origin isolates. J Virol 81, 1264112653.

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Kolakofsky, D., Roux, L., Garcin, D. and Ruigrok, R. W. H. (2005). Paramyxovirus mRNA editing, the ‘rule of six’ and error catastrophe: a hypothesis. J Gen Virol 86, 1869-1877. Kopylova, E., Noe, L. and Touzet, H. (2012). SortMeRNA: fast and accurate filtering of ribosomal RNAs in metatranscriptomic data. Bioinformatics 28, 3211-3217. Li, H. and Durbin, R. (2010). Fast and accurate long-read alignment with Burrows-Wheeler transform. Bioinformatics 26, 589-595. Li, H., Handsaker, B., Wysoker, A., Fennell, T., Ruan, J., Homer, N., Marth, G., Abecasis, G., Durbin, R. and Genome Project Data Processing, S. (2009). The Sequence Alignment/Map format and SAMtools. Bioinformatics 25, 2078-2079. Miller, P. J., Afonso, C. L., El Attrache, J., Dorsey, K. M., Courtney, S. C., Guo, Z. J. and Kapczynski, D. R. (2013). Effects of Newcastle disease virus vaccine antibodies on the shedding and transmission of challenge viruses. Dev Comp Immunol 41, 505-513. Miller, P. J., Decanini, E. L. and Afonso, C. L. (2010). Newcastle disease: Evolution of genotypes and the related diagnostic challenges. Infect Genet Evol 10, 26-35. Miller, P. J., Estevez, C., Yu, Q. Z., Suarez, D. L. and King, D. J. (2009). Comparison of Viral Shedding Following Vaccination with Inactivated and Live Newcastle Disease Vaccines Formulated with Wild-Type and Recombinant Viruses. Avian Dis 53, 39-49. Miller, P. J., King, D. J., Afonso, C. L. and Suarez, D. L. (2007). Antigenic differences among Newcastle disease virus strains of different genotypes used in vaccine formulation affect viral shedding after a virulent challenge. Vaccine 25, 7238-7246. Munir, M., Abbas, M., Khan, M. T., Zohari, S. and Berg, M. (2012a). Genomic and biological characterization of a velogenic Newcastle disease virus isolated from a healthy backyard poultry flock in 2010. Virol J9. Munir, M., Cortey, M., Abbas, M., Qureshi, Z. U., Afzal, F., Shabbir, M. Z., Khan, M. T., Ahmed, S., Ahmad, S., Baule, C., Stahl, K., Zohari, S. and Berg, M. (2012b). Biological characterization and phylogenetic analysis of a novel genetic group of Newcastle disease virus isolated from outbreaks in commercial poultry and from backyard poultry flocks in Pakistan. Infection Genetics and Evolution 12, 1010-1019. Munir, M., Shabbir, M. Z., Yaqub, T., Shabbir, M. A. B., Mukhtar, N., Khan, M. R. and Berg, M. (2012c). Complete Genome Sequence of a Velogenic Neurotropic Avian Paramyxovirus 1 Isolated from Peacocks (Pavo cristatus) in a Wildlife Park in Pakistan. JVirol 86, 1311313114. Perozo, F., Merino, R., Afonso, C. L., Villegas, P. and Calderon, N. (2008). Biological and phylogenetic characterization of virulent Newcastle disease virus circulating in Mexico. Avian Dis 52, 472-479. Rehmani, S. F., Wajid, A., Bibi, T., Nazir, B., Mukhtar, N., Hussain, A., Lone, N. A., Yaqub, T. and Afonso, C. L. (2015). Presence of virulent Newcastle disease virus in vaccinated chickens in farms in Pakistan. J Clin Microbiol 53, 1715-1718. Romer-Oberdorfer, A., Veits, J., Werner, O. and Mettenleiter, T. C. (2006). Enhancement of pathogenicity of Newcastle disease virus by alteration of specific amino acid residues in the surface glycoproteins F and HN. Avian Dis 50, 259-263. Ronquist, F. and Huelsenbeck, J. P. (2003). MrBayes 3: Bayesian phylogenetic inference under mixed models. Bioinformatics 19, 1572-1574. Samuel, A., Nayak, B., Paldurai, A., Xiao, S., Aplogan, G. L., Awoume, K. A., Webby, R. J., Ducatez, M. F., Collins, P. L. and Samal, S. K. (2013). Phylogenetic and pathotypic characterization of newcastle disease viruses circulating in west Africa and efficacy of a current vaccine. J Clin Microbiol 51, 771-781. Shabbir, M. Z., Abbas, M., Yaqub, T., Mukhtar, N., Subhani, A., Habib, H., Sohail, M. U. and Munir, M. (2013a). Genetic analysis of Newcastle disease virus from Punjab, Pakistan. Virus Genes 46, 309-315.

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Shabbir, M. Z., Goraya, M. U., Abbas, M., Yaqub, T., Shabbir, M. A., Ahmad, A., Anees, M. and Munir, M. (2012). Complete genome sequencing of a velogenic viscerotropic avian paramyxovirus 1 isolated from pheasants (Pucrasia macrolopha) in Lahore, Pakistan. J Virol 86, 13828-13829. Shabbir, M. Z., Zohari, S., Yaqub, T., Nazir, J., Shabbir, M. A., Mukhtar, N., Shafee, M., Sajid, M., Anees, M., Abbas, M., Khan, M. T., Ali, A. A., Ghafoor, A., Ahad, A., Channa, A. A., Anjum, A. A., Hussain, N., Ahmad, A., Goraya, M. U., Iqbal, Z., Khan, S. A., Aslam, H. B., Zehra, K., Sohail, M. U., Yaqub, W., Ahmad, N., Berg, M. and Munir, M. (2013b). Genetic diversity of Newcastle disease virus in Pakistan: a countrywide perspective. Virol J 10, 170. Siddique, N., Naeem, K., Abbas, M. A., Ali Malik, A., Rashid, F., Rafique, S., Ghafar, A. and Rehman, A. (2013). Sequence and phylogenetic analysis of virulent Newcastle disease virus isolates from Pakistan during 2009-2013 reveals circulation of new sub genotype. Virology 444, 37-40. Snoeck, C. J., Ducatez, M. F., Owoade, A. A., Faleke, O. O., Alkali, B. R., Tahita, M. C., Tarnagda, Z., Ouedraogo, J. B., Maikano, I., Mbah, P. O., Kremer, J. R. and Muller, C. P. (2009). Newcastle disease virus in West Africa: new virulent strains identified in noncommercial farms. Arch Virol 154, 47-54. Tamura, K., Stecher, G., Peterson, D., Filipski, A. and Kumar, S. (2013). MEGA6: Molecular Evolutionary Genetics Analysis version 6.0. Mol Biol Evol 30, 2725-2729. Tirumurugaan, K. G., Kapgate, S., Vinupriya, M. K., Vijayarani, K., Kumanan, K. and Elankumaran, S. (2011). Genotypic and pathotypic characterization of Newcastle disease viruses from India. PLoS One6, e28414. Tsai, H. J., Chang, K. H., Tseng, C. H., Frost, K. M., Manvell, R. J. and Alexander, D. J. (2004). Antigenic and genotypical characterization of Newcastle disease viruses isolated in Taiwan between 1969 and 1996. Vet Microbiol 104, 19-30. Ujvari, D., Wehmann, E., Kaleta, E.F., Werner, O., Savic, V., Nagy, E., Czifra, G. and Lomniczi, B. (2003). Phylogenetic analysis reveals extensive evolution of avian paramyxovirus type 1 strains of pigeons (C. livia) and suggests multiple species transmission. Virus Res 96, 63-73. Umali, D. V., Ito, H., Suzuki, T., Shirota, K., Katoh, H. and Ito, T. (2013). Molecular epidemiology of Newcastle disease virus isolates from vaccinated commercial poultry farms in non-epidemic areas of Japan. Virol J 10, 330. Wang, J., Liu, H., Liu, W., Zheng, D., Zhao, Y., Li, Y., Wang, Y., Ge, S., Lv, Y., Zuo, Y., Yu, S. and Wang, Z. (2015). Genomic characterizations of six pigeon paramyxovirus type 1 viruses isolated from live bird markets in China during 2011 to 2013. PLoS One 10, e0124261. Wise, M. G., Suarez, D. L., Seal, B. S., Pedersen, J. C., Senne, D. A., King, D. J., Kapczynski, D. R. and Spackman, E. (2004). Development of a real-time reverse-transcription PCR for detection of newcastle disease virus RNA in clinical samples. J Clin Microbiol 42, 329-338. Yang, C. Y., Shieh, H. K., Lin, Y. L. and Chang, P. C. (1999). Newcastle disease virus isolated from recent outbreaks in Taiwan phylogenetically related to viruses (genotype VII) from recent outbreaks in western Europe. Avian Dis 43, 125-130. Yu, L., Wang, Z., Jiang, Y., Chang, L. and Kwang, J. (2001). Characterization of newly emerging Newcastle disease virus isolates from the People’s Republic of China and Taiwan. J Clin Microbiol 39, 3512-3519. Yurchenko, K. S., Sivay, M. V., Glushchenko, A. V., Alkhovsky, S. V., Shchetinin, A. M., Shchelkanov, M. Y. and Shestopalov, A. M. (2015). Complete Genome Sequence of a Newcastle Disease Virus Isolated from a Rock Dove (Columba livia) in the Russian Federation. Genome Announc 3. Zhang, S., Wang, X., Zhao, C., Liu, D., Hu, Y., Zhao, J. and Zhang, G. (2011). Phylogenetic and pathotypical analysis of two virulent Newcastle disease viruses isolated from domestic ducks in China. PLoS One6, e25000.

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Bluetongue in India: outbreaks to vaccine outcome Minakshi Prasad, Koushlesh Ranjan1 and Gaya Prasad1 Department of Animal Biotechnology, LLR University of Veterinary and Animal Sciences, Hisar 1 SVP University of Agriculture and Technology, Meerut

Introduction Bluetongue (BT) is one of the important viral diseases of domestic and wild ruminants in India. It is transmitted by the Culicoides (bitting midges) vector. The disease is caused by non-enveloped icosahedral Bluetongue virus (BTV), which belongs to the genus Orbivirus and family Reoviridae. It can infect all species of ruminants especially small ruminants such as sheep (Maclachlan and Guthrie, 2010). The clinical signs of BTV infection are more severe and confined to mostly whitetailed deer and sheep (Darpel et al., 2007). The cattle or buffalo are clinically not affected by BT. They act as reservoir for the disease. Exceptionally, however, BTV8 was reported from clinically infected cattle. BT is listed under the category of multiple species diseases by Office International des Epizooties (OIE, 2013). Globally, there are twenty seven distinct BTV serotypes have been reported (Jenckel et al., 2015). The segmented nature of BTV genome allows the reassortment of genome segments, especially when mammalian host or Culicoides vector is simultaneously infected by two or more different BTV serotypes. This led to the evolution of new isolates and serotypes of BTV. The Indian subcontinent lies between 8.4°N and 37.6°N and 68.7°E and 97.25°E. The overall climate of India is hot and humid. The monsoon enters the India first in southern region during late May or early June and reaches to north later on. Thus, June to October is the months of rain-bearing monsoon. The monsoon season is favorable to various insect vectors (including Culicoides) and infectious diseases (like BT) transmitted by them. The several species of domestic and wild ruminants of India are known to be significantly susceptible to BTV infection. The large population of susceptible animals along with favorable climatic conditions made India endemic for BT. Economic impact of bluetongue in India The BT disease has a potential to spread rapidly. Thus, it creates one of the major barriers in international trade of animals and its products. It causes economic losses in terms of high morbidity, mortality, abortion, fetal death and deformities as well as milk, meat and fleece losses. On average 2%-30% of the animals infected by BT die. However, the number may reach up to 100% in highly susceptible sheep. BT caused death of 300,000 sheep and goats in Tamil Nadu during the monsoon season of 1997-1998 (Ilango, 2006). It is the major contributing factor for annual economic losses in Indian sheep industry. During 1991-2005, BT caused maximum economic losses (60.8%) among all infectious diseases in sheep. Due to its possible socio-

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economic consequences, mandatory export restrictions and surveillance requirements are imposed on movement of live animals and its products, germplasm, embryo from BT-endemic to BT-free countries (Velthuis et al., 2009). BT affects particularly native sheep population and causes high mortality, overall loss of productivity, weight loss and wool break. Thus it causes significant economic losses to less-affluent farming community, because native sheep is mainly reared by these farmers. Once BTV established in a particular area, it is very difficult to completely eradicate. Bluetongue Epidemiology in India First phase of bluetongue The exotic breeds of sheep were imported from USA and South Africa to India during 1960s and 1970s for the genetic improvement of native breeds by crossbreeding. The BT disease was travelled to India along with live animals imported. BT was first reported from Indian subcontinent in 1964 (Sapre, 1964). During 1967 to 1970 the disease was reported in exotic sheep, namely Southdown, Rambouillet, Russian Merino and Corriedale. Severe BT was also reported in the Dorset breed in Andhra Pradesh in 1974. However, the disease symptoms were not reported from the native sheep maintained in close proximity with infected ones. Later on, the disease was recorded in native sheep population from several parts of India. Since 1981, annual BT outbreaks have been reported. Although first evidence of BT was seen in India in 1964, BTV could not be isolated from affected animals. The first BTV serotypes to be isolated from affected sheep were serotypes 8 and 18. Subsequently, serotype 1 was isolated from Rambouillet sheep from Central Sheep Breeding Farm (CSBF), Hisar (Jain et al., 1986). Later on, many other workers reported isolation of different BTV serotypes (BTV2, 3, 9, 16, 18 and 23) from different parts of the country (Prasad et al., 1994). The presence of BTV-specific antibodies in animals is a major evidence for occurrence of BTV infection. During 1981, BT was widely spread in southern India. The disease was initially detected in Karnataka and in the adjoining regions of Maharashtra and Andhra Pradesh, with mortality rates ranging from 2% to 50%. A cyclic pattern of the disease was observed with variations in the severity of infection. Later on, a severe form of BT infection was reported with an overall morbidity, mortality and case fatality rate of 32%, 8% and 25%, respectively, in rural areas of Maharashtra (Kulkarni et al., 1992). The study of BT outbreak pattern in organized farms and rural flocks of Andhra Pradesh state revealed that the pattern of the disease in organized farms and rural flocks is quite different. The higher morbidity and mortality in rural areas was attributed to stress factors, such as poor nutrition, parasitic burden, fatigue due to long walks and absence of veterinary facility. The BT occurrence in different regions of India varies according to time of rainfall. Maximal numbers of outbreaks were recorded during the north-east monsoon period (October to December) followed by the south-west monsoon period (June to September). In the Erode District of Tamil Nadu a severe outbreak of BT disease was observed during the month of heavy rainfall (November and December).

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Recent bluetongue outbreak in India The BT outbreaks have been reported from sheep and goat in several states of India. India being endemic for BT, 21 different serotypes of BTV have been reported (Prasad et al., 2009). Recently, however, the 22nd serotype, i.e. BTV21, has been isolated (Susmitha et al., 2012). The serological survey on BTV has indicated the presence of BTV-specific antibodies in Indian sheep, goat, cattle, buffalos, camels and several wild ruminants (Reddy et al., 2016). Several serotypes of BTV have been isolated from the regions where sheep population is higher. The north-western region of the country is mostly affected with BTV1. The BTV1 has been isolated from sheep, goat and Culicoides vector in this region (Prasad et al., 1994). BTV23 has been reported from Uttarakhand, Madhya Pradesh, Maharashtra, Karnataka, Jammu and Kashmir (Tembhurne et al., 2010). The BTV-specific antibodies in small ruminant have been reported from Delhi. The Agar Gel Immunodiffusion test (AGID) test showed an overall seropositivity of 13.21% for BTV in non-descript goat from Delhi. During 2005-2009, the serotype-specific serum-neutralization test for BTV in Andhra Pradesh showed 50.0, 44.23, 21.15, 26.92 and 15.38% seroprevalence of BTV serotypes 1, 2, 9, 10 and 23, respectively. The BTV2, BTV9 and BTV15 have been reported from sheep in the Andhra Pradesh state. The serological survey using dot ELISA and competitive ELISA (C-ELISA) techniques confirmed the BTV prevalence in Kerala state. The overall BT seroprevalence has increased to 9.3% in domestic ruminants in Kerala. High seroprevalence of bluetongue in sheep, goat and cattle has also been reported in the northeast region (Assam). However, the live virus could not be isolated (Joardar et al., 2013). The serological study also confirmed the high prevalence (86%) of BTV in Mithun (Bos frontalis) in Northeast region (Nagaland state) of India. The serotypes BTV15 and BTV21 have been isolated from sheep in eastern part of India (West Bengal). Globally, all BT viruses can be divided into eastern and western topotype based on geographic origin of the virus. Both eastern and western topotypes of BTV have been isolated from different region of India. The trade of livestock and their products along with live attenuated vaccine from western countries has played a major role in occurrence of western BTV genome segments in India. The eastern topotype BTV1 and western topotype BTV2 and BTV10 have been reported recently. The complete genome sequence of a reassortant strain of BTV16 of goat origin from India has been obtained recently (Minakshi et al., 2012). Moreover, BTV serotype 1 from Culicoides oxystoma vector in Gujarat state and serotype 10 from Andhra Pradesh have been isolated (Dadawala et al., 2012). The Indian isolates of BTV show a great degree of reassortment between different serotypes and also between eastern and western topotype viruses within same serotype. Multiple serotypes of BT viruses are circulating in a geographical area, which leads to infection of individual animals and herds with more than one serotype. It facilitates the exchange of genetic material between different viruses and evolution of new reassortant strains of BTV (Reddy et al., 2015). A reassortant

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strain of BTV16 having segment 5 (ns1 gene) of western origin and other segments of eastern origin has been reported from Andhra Pradesh state (Kumar et al., 2013). BTV vaccines in India Vaccination can reduce the economic losses by decreasing animal mortality and makes safe transfer and trading of animals and their products from one country to other. Vaccination may prevent the clinical form of BT (Bhanuprakash et al., 2009). Ideally, vaccination should be against all 27 serotypes because of very low cross-protection between different serotypes. Therefore, BT vaccines should be serotype-specific. Thus, before the use of BT vaccine in an area, the serotype prevalent in that area should be taken into account (Bhanuprakash et al., 2009). Furthermore, there is a serious drawback in terms of availability of safe vaccines for BTV. Multivalent live vaccines (MLV) have been used to control BT in several countries, such as in South Africa, USA, Israel and several parts of Europe (Savini et al., 2008). However, MLV cannot be considered as safe for vaccination. MLV viruses may either revert back to virulent strain or reassort with other field viruses. Thus, it may be a reason for spread of vaccine-like viruses from vaccinated to unvaccinated animals, which are associated with serious BT outbreaks (Savini et al., 2008; Gollapalli et al., 2012). Moreover, MLV vaccines can also cross the placenta to infect the fetus and may lead to abortions, stillbirths and fetal abnormalities in cattle (Maclachlan and Osburn, 2008; Savini et al., 2008). In India, sheep and goat are reared mostly by small farmer or nomadic herdsmen. Therefore, vaccination with MLV is not possible throughout the country. Moreover, it may result in spread of MLV-like virus in unvaccinated flocks or its reassortant virus with previously persisting field virus, causing serious BT outbreak in animals. Due to serious side effects of MLV, inactivated vaccines against BTV may be a choice. It was used in Europe against BTV8 outbreak. Some of the inactivated BTV vaccine preparations were also produced in India. The binary ethylenimine (BEI)-inactivated saponified vaccines for BTV1 and BTV18 were used earlier, which elicited appreciable immune response post-vaccination in Indian sheep (Pandey et al., 2006). The BEI-inactivated BTV1 vaccine induced appreciable cellmediated immune (CMI) response against heterologous virulent strain of BTV23 in Indian native sheep (Umeshappa et al., 2010). Other viral inactivating agenthydroxylamine-was also used for preparation of inactivated vaccine in India. An inactivated vaccine for BTV18 was prepared using hydroxylamine as inactivating agent and Al (OH)3 gel/saponin as adjuvant for immunization of Indian sheep. The vaccine induced group-specific non-neutralizing antibodies to BTV18 as soon as seven days post-vaccination and reduced the duration of viremia during next challenge with homotypic virus dose (Ramakrishnan et al., 2005). Recently, a pentavalent inactivated adjuvant vaccine containing BTV serotypes 1, 2, 10, 16 and 23 has been developed under AINP-BT of ICAR (Reddy et al., 2010, Annual reports AINP-BT, ICAR 2012-14). The vaccine technology has been transferred to IIL, Hyderabad and is commercially produced in the name of ‘Raksha Blu’ is available

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commercially in India. However, inactivated vaccines have relatively slow onset of immunity and are commercially unavailable for most of the serotypes. Now, modern molecular-technology based new generation vaccines such as recombinant canarypox virus expressing VP2 and VP5 proteins and baculovirusexpressed virus-like particles (VLPs) have been developed. However, they are not very successful mainly due to the cost effectiveness (Boone et al., 2007; Noad and Roy, 2009). Other approaches, such as subunit vaccination against VP2 protein, have also been explored, but expression of immunogenic VP2 is a challenge because the specific conformation of individual epitopes (DeMaula et al., 2000). References Bhanuprakash V, Indrani BK, Hosamani M, Balamurugan V and Singh RK (2009). Bluetongue vaccines: the past, present and future. Expert Reviews Vaccines 8, 191-204. Boone JD, Balasuriya UB, Karaca K, Audonnet JC, Yao J, He L, Nordgren R, Monaco F, Savini G, Gardner IA and Maclachlan NJ (2007). Recombinant canarypox virus vaccine coexpressing genes encoding the VP2 and VP5 outer capsid proteins of bluetongue virus induces high level protection in sheep. Vaccine, 25, 672-678. Dadawala AI, Biswas SK, Rehman W, Chand K, De A, Mathapati BS, Kumar P, Chauhan HC, Chandel, BS and Mondal B (2012). Isolation of bluetongue virus serotype 1 from Culicoides vector captured in livestock farms and sequence analysis of the viral genome segment-2. Transbound. Emerg. Dis. 59, 361-368. DeMaula CD, Bonneau KR and Maclachlan NJ (2000). Changes in the outer capsid proteins of bluetongue virus serotype 10 that abrogate neutralization by monoclonal antibodies. Virus Res. 67, 59-66. Gollapalli SR, Mallavarapu S, Uma M, Rao PP, Susmitha B, Prasad PU, Chaitanya P, Prasad G, Hegde NR and Reddy YN (2012). Sequences of genes encoding type-specific and groupspecific antigens of an Indian isolate of bluetongue virus serotype 10 (BTV-10) and implications for their origin. Transbound. Emerg. Dis. 59, 165-172. Ilango K (2006). Bluetongue virus outbreak in Tamil Nadu, southern India: Need to study the Indian biting midge vectors, Culicoides Latreille (Diptera: Ceratopogonidae). Current Science. 90(2), 163-167. Jain NC, Sharma R and Prasad G (1986). Isolation of bluetongue virus from sheep in India. Vet. Rec. 119, 17-18. Joardar SN, Barkataki B, Halder A, Lodh C and Sarma D (2013). Seroprevalence of bluetongue in north eastern Indian state-Assam. Vet. World. 6(4), 196-199. Kulkarni DD, Bannalikar AS, Karpe AG, Gujar MB and Kulkarni MN (1992). Epidemiological observations on bluetongue in sheep in Marathawada region of Maharashtra State in India. In Bluetongue, African horse sickness and related orbiviruses (T.E. Walton & B.I. Osburn, eds). Proc. Second International Symposium, Paris, 17-21 June 1991. CRC Press, Boca Raton, pp. 85. Kumar P, Minakshi P, Ranjan K, Dalal R and Prasad G (2013). Evidence of reassortment between eastern and western topotype strains of bluetongue virus serotype 16 (BTV-16) from India. Adv. Anim. Vet. Sci. 1 (4S), 14-19. Maclachlan NJ and Osburn BI (2008). Induced brain lesions in calves infected with bluetongue virus. Vet. Rec. 162, 490-491.

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Minakshi P, Singh R, Ranjan K, Kumar P, Joshi CG, Reddy YKM and Prasad G (2012). Complete Genome Sequence of Bluetongue Virus Serotype 16 of Goat Origin from India. J. Virol. 86 (15), 8337. Noad R and Roy P (2009). Bluetongue vaccines. Vaccine 27 (suppl.4), D86-D89. Pandey AB, Nandi S, Dubey SC, Sonawane GG, Mondal B, Bhanuprakash V, Audarya SD, Suresh I, Sharma K, Prasad G, Singh RK, Singh N and Chauhan RS (2006). Trial of inactivated bluetongue vaccine in Bharat-Merino sheep. J. Immuno. Immunopatho. 8(2), 154-146. Prasad G, Garg AK, Minakshi, Kakker NK and Srivastava RN (1994). Isolation of bluetongue virus from sheep in Rajasthan state. Rev. Sci. Tech. 13, 935-938. Prasad G, Sreenivasulu D, Singh KP, Mertens PPC and Maan S (2009). Bluetongue in the Indian subcontinent. In: Bluetongue. (Eds. Mellor P, Baylis M and Merten P C). Elsevier Ltd., London. Pp. 167-195. Ramakrishnan MA, Pandey AB, Singh KP, Singh R and Mehrotra ML (2005). Immune response and protective efficacy in sheep immunized with hydroxylamine-inactivated bluetongue virus vaccine. Vet. Italiana. 41 (3), 149-155. Reddy YKM, Brindha K., Ganesan P.I., Srinivas K., Reddy G.S. and Minakshi P. (2016). Occurrence of Bluetongue in ruminants in Tamil Nadu, South India. Veterinaria Italiana 2016, 52 (3), xxx-xxx. doi: 10.12834/VetIt.502.2421.2 Impact factor 0.675 NAAS 6.68/V010 Reddy YKM, Manohar BM, Pandey AB, Reddy YN, Prasad G and Chauhan RS (2010). Development and evaluation of inactivated pentavalent adjuvanted vaccine for Bluetongue. Indian Vet. J. 87, 434-436. Reddy YV, Krishnajyothi Y, Susmitha B, Devi BV, Brundavanam Y, Gollapalli SR, Karunasri N, Sonali B, Kavitha K, Patil SR, Sunitha G, Putty K, Reddy GH, Reddy YN, Hegde NR, Rao PP (2015). Molecular Typing of Bluetongue Viruses Isolated Over a Decade in South India. Transbound Emerg Dis. doi: 10.1111/tbed.12320. Sapre SN (1964). An outbreak of bluetongue in goats and sheep. Ind. Vet. Rev. 15,78-80. Savini G, MacLachlan NJ, Sanchez-Vizcaino JM, Zientara S (2008). Vaccines against bluetongue in Europe. Comp. Immuno. Microbiol. Infect. Dis. 31, 101-120. Susmitha B, Sudheer D, Rao PP, Uma M, Prasad G, Minakshi P, Hegde NR, Reddy YN (2012). Evidence of bluetongue virus serotype 21 (BTV-21) divergence. Virus Genes. 44, 466-469. Tembhurne PA, Mondal B, Pathak KB, Biswas SK, Sanyal A, Yadav MP, Bandyopadhyay SK, Singh RK (2010). Segment-2 sequence analysis and cross neutralization studies on some Indian bluetongue viruses suggest isolates are VP2-variants of serotype 23. Arch. Virol. 155, 89-95. Umeshappa CS, Singh KP, Pandey AB, Singh RP, Nanjundappa RH (2010). Cell-mediated immune response and cross-protective efficacy of binary ethylenimine-inactivated bluetongue virus serotype-1 vaccine in sheep. Vaccine. 28(13), 2522-2531. Velthuis AG, Saatkamp HW, Mourits MC, de Koeijer AA, and Elbers AR (2009). Financial consequences of the Dutch bluetongue serotype 8 epidemics of 2006 and 2007. Prev. Vet. Med. 93, 294-304.

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Brief overview: bacterial diseases of equines 1

S.K. Khurana1 and Kuldeep Dhama2

National Research Centre on Equines, Sirsa Road, Hisar, Haryana, India Department of Pathology, Indian Veterinary Research Institute, Izatnagar, Bareilly, U.P., India

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Introduction Bacterial pathogens play very important role in equines. The bacterial diseases of equines affect different age groups i.e. neonates, young or adults. Different diseases have variable effect starting from morbidity to mortality. Some diseases like glanders and strangles have been eradicated from several developed countries especially from the temperate regions. The pathogenesis of several diseases like Rhodococcus equi and Actinobacillus equilli infections is not fully understood; moreover their rapid and sensitive diagnostic tools are also not available, thus making their prevention and control very difficult. Diagnostic laboratories generally apply conventional cultural and serological methods for bacterial disease diagnosis. The accurate identification of organism to the species level is essential for epidemiological, preventive and control purposes, so molecular techniques are used for reliable and rapid identification. Here, we are providing information regarding some of the bacterial diseases which are important in equines. 1.

Strangles and other streptococcal infections

Strangles is characterized by abscessation of lymph nodes of head and and neck and is caused by β-haemolytic Lancefield group C streptococcal bacteria Streptococcus equi subspecies equi. However, the infections caused by Streptococcus equi subspecies zooepidemicus are also very common. This subspecies is present as resident microflora on skin, nasopharynx, gastro-intestinal tract and vagina of healthy equines and is isolated as the only pathogen in disease conditions like abortions, endometritis, cervicitis, pneumonia, abscesses, joint infections and others. The equine Lancefield group C streptococci are differentiated biochemically based on their ability to ferment sorbitol, lactose and trehalose (Quinn et al., 1994). Further, Polymerase Chain Reaction (PCR) genetically identifies different species and subspecies (Alber et al., 2004; Baverud et al., 2007; Preziuso et al., 2010;). Strangles is caused by nasopharyngeal infection by Streptococcus equi and spreads rapidly to the lymph nodes of the head. Multiplication of S. equi and early lymphadenitis proceeds. There is massive infiltration of polymorphonuclear leukocytes into the lymph nodes, until an abscess capsule forms along with a sinus tract to drain pus. This process last for 2-3 weeks and is associated with depression, loss of appetite, fever, mucopurulent nasal discharge and dyspnea. Abscesses can form in other body organs and their rupture is fatal in up to 10% of cases, this form is also known as “bastard strangles”. Purpura hemorrhagica (petechial haemorrhage

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associated with oedema of the limbs, eye lids and gums) may occur in association with circulating antibody complexes with S. equi M-like protein. The peripheral oedema can be so extreme that circulatory failure and death may follow. Streptococcus equi subspecies zooepidemicus infection S. equi subspecies zooepidemicus is β-haemolytic streptococci in Lancefield group C. Streptococcus equi subspecies zooepidemicus is considered opportunistic pathogen in horse, but causes infection in cattle, sheep, goat, pig and dog also. S. equi subspecies zooepidemicus has more than 98% DNA sequence homology with S. equi subspecies equi. Pelkonen et al. (2013) have shown that S. equi subspecies zooepidemicus is transmitted from horse to human beings and they found that human and equine isolates were identical or closely related. Lindahl et al. (2013) reported an outbreak of repiratory disease due to S. equi subspecies equi. Downar et al. (2001) have documented infection of Streptococcal meningitis from close contact with an infected horse. 2.

Rhodococcus equi infection

Respiratory diseases are common in young horses, especially in foals between one and six months of age. The major causes of pneumonia in foals are bacterial in nature, among which Rhodocococcus equi and Streptococcus zooepidemicus are the most important. Several other aerobic bacterial species may also be involved including Klebsiella pneumonia, Staphylococcus spp., Bordetella bronchiseptica, Escherichia coli, Pasteurella spp., Pseudomonas spp.,Actinobacillus spp and Salmonella spp. Among all cases of bacterial foal pneumonia Rhodocococcus equi appears to be most important. Rhodococcus equi is a gram positive, pleomorphic, coccobacillus, aerobic soil actinomycete responsible primarily for severe respiratory disease of young foals with high mortality rate (Prescott, 1991; Yager et al., 1991; Khurana et al., 2009; Giguere et al., 2011a,b; Khurana, 2014; Khurana et al., 2014; Khurana, 2015; Khurana et al., 2015; Chhabra et al., 2015; Chhabra et al., 2016). It is a facultative intracellular pathogen of macrophages. R. equi also causes extra-pulmonary complications in equines including enteritis, arthritis and abscesses in abdomen (Giguere and Prescott, 1997). R. equi was first recovered from lung of a foal as Corynebacterium equi (Magnusson, 1923) and was reclassified as R. equi (Goodfellow and Alderson, 1977). This organism is emerging as an important pathogen in AIDS patients (Weinstock and Brown, 2002), drug therapy (Mizuno et al., 2005) and some other immunosuppressive conditions (Napoleao et al., 2005). The most common manifestation of human R. equi infections is pneumonia, others include fever, diarrhoea, abscesses in various internal organs and arthritis. Although Rhodocococcus equi is found in the soil of most farms, pneumonia caused by this organism can be endemic, sporadic or unrecognized depending upon the farm studied. Several factors may influence the incidence of the disease including the degree of contamination on the farm, density of horses, climatic conditions and virulence of the isolate. The infection is acquired through inhalation.

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Morbidity rates of 5-17% with mortality rates of 40-80% have been reported by Elissalde et al. (1980). Farms that have endemic problems with Rhodocococcus equi are usually contaminated with a higher proportion of virulent strains. The key to the pathogenesis of Rhodocococcus equi is related to the ability of the organism to survive and replicate within alveolar macrophages by inhibiting phagosomelysosome fusion after phagocytosis (Hietala et al., 1987 ; Zink et al., 1987). Virulence associated protein A (Vap A), a cell surface lipoprotein is essentially required for virulence in foals whereas virulence associated protein B (Vap B) is often associated with disease in human beings and pigs. Intracellular localization of R. equi is responsible for its prolonged and difficult therapeutic management. No suitable serodiagnostic test or vaccination is available for R. equi infection of equines as well as humans till date (Khurana, 2015). Human beings acquire infection mainly through inhalation of dust harboring bacteria, from domestic animals including equines and wound, however man to man transmission is thought to be rare (Weinstock and Brown, 2002). R. equi infections are conventionally diagnosed by culturing and subsequent phenotypic analysis of the isolates by means of classical morphological and biochemical tests. However, the colony characteristics, cellular morphology, and reaction to acid-fast staining differ between R. equi isolates.Identification of R. equi by classical bacteriological techniques is sometimes difficult and time consuming, and misclassification of an isolate is not uncommon. Agar gel diffusion test developed by Nakazawa et al. (1987) and ELISA by Giguere et al. (2003) have not been found to be of much promise for diagnosis of R. equi serologically. In India, diagnosis through post-mortem examination (Garg et al., 1985; Saxena and Narwal, 2009) and isolation of R. equi from clinical samples (Khurana et al., 2009). Various PCR assays have been developed (Sellon et al., 2001; Arriaga et al., 2002; Ladron et al., 2003; Oldfield et al., 2004; Ocampo-Sosa et al., 2007; Pusterla et al., 2007; Letek et al., 2008; Monego et al., 2009; Chhabra et al., 2015; Khurana et al., 2015). The most valuable diagnostic procedures are combination of cultural methods along with PCR assay (Khurana, 2015). Rifampicin along with macrolides is drug of choice for treatment of R. equi infection. Rifampicin resistance have been reported which is posing a challenge in therapy (Asoh et al., 2013; Burton et al., 2013; Goldstein, 2014; Liu et al., 2014). Chhabra et al. (2016) have studied the resistotypes of R. equi isolates isolated from foals with respiratory problems which could be useful in deciding treatment regimen. Proper management and sanitation at farms is very important for control of disease at equine farms. Hygiene is important in immunocompromised human beings. 3.

Glanders and melioidosis

Glanders is a contagious and fatal disease of horse, mules and donkey with zoonotic potential (Malik et al., 2010; Varga et al., 2012; Verma et al., 2014). This 28 

 

is a disease known since ancient times and was identified in 4th century BC by Hippocrates (Colahan et al., 1999). The disease is caused by a non-motile, non-spore forming gram negative bacillus called Burkholderia mallei. This was previously classified variously as Pfeifferella, Loefflerella, Malleomyces or Actinobacillus). Most common mode of transmission of this organism is inhalation and ingestion of contaminated feed and water. The disease occurs in chronic form in horses, where bacteria are found in nasal discharges and skin lesions (OIE 2004, 2008), whereas in mules and donkeys the disease occurs in acute form (Hunting, 1913; Gulati and Gautam, 1962). The acute form of glanders involves pulmonary, cutaneous and nasal sites (Jubb et al., 1993), and is characterized by pyrexia, cough, discharges from nostrils, ulcers on nasal mucosa and nodules on the skin finally leading to death. In most outbreaks, these forms are not clearly distinct and may occur simultaneously in an animal. Chronic form in horse also shows all three clinical manifestations which include pulmonary, nasal and cutaneous forms. The human glanders is not very common, but it is having very high mortality rate of 90-95% in untreated septicemic infection and 50% in treated humans. Human outbreaks have not been reported. Guinea-pigs are highly susceptible, and males are used for testing of potentially infected material. Intraperitoneal injections are given to attempt to elicit the Strauss reaction (orchitis). The mallein test is a sensitive and specific clinical test for glanders. This organism is very important from biological warfare angle due to high rate of mortality and ability of small number of organisms to establish the infection. The diagnosis of glanders is done by isolation and allergic test (mallein test). The test is not very specific, so it should be used along with complement fixation test which is also prescribed for international trade. The test is not very specific, so it should be used along with complement fixation test (CFT), which is also prescribed for international trade. Various serological tests for diagnosis of glanders include complement fixation test (CFT), indirect haemagglutination assay (IHA) and ELISA. Singha et al. (2014) have developed a indirect ELISA using truncated TssB protein for serodiagnosis of glanders. Various PCR assays have been developed (Grishkina and Samygi, 2010; Zhang et al., 2012) which have been found to be rapid, sensitive and specific. Janse et al. (2013) developed a multiplex qPCR for detection and differentiation of B.mallei and B.pseudomallei (meliodiosis agent). B. mallei and B. pseudomallei have been reported to manifest high similarity with regard to clinical syndromes, phenotypic and genotypic characters and found serologically indistinguishable due to cross reactions. Molecular diagnosis based on genome of these organisms has solved this problem. A PCR for the specific detection of B. mallei DNA has been developed that allows differentiation between B. mallei and B. pseudomallei.

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Diagnosis in human beings is generally done by CFT and imaging studies. No vaccine is available for prevention of glanders for animals or humans (Burtnick et al., 2012). In case of death suspected due to glanders the carcass should not be opened and must be buried deep or incinerated. 4.

Salmonella Abortus equi and other salmonellosis

Salmonella enterica serovar Abortus equi is a important cause of abortions and reproductive problems in equines. The abortions are seen during last quarter of pregnancy. It is also associated with joint ill, bronchopneumonia and polyarthritis in foals and bursitis, fistulous withers and testicular infection in stallion. The role of infected stallions in the transmission of infection to mares through coitus has been demonstrated. In India, it was first reported among mares maintained at Government Livestock Farm, Hisar in 1919. Diagnosis is conventionally done by isolation and identification of organism by cultural methods in aborted material/ vaginal/ uterine or prepucial swabs. Serology is done by Standard tube agglutination test. Mitterer et al (2004) have developed a Microarray-Based identification system of Salmonella enterica serovar Abortus equi in clinical samples by solid-phase PCR amplification of 23S ribosomal DNA sequences. Control of disease is based on the use of immunoprophylactic agents. Most commonly a bacterin prepared from formalized S. Abortus equi cells is being used. Salmonellosis caused by other species of Salmonella affect humans, horses, most mammals, and birds. It can cause debilitating diarrhea which may lead to mortality. Salmonella can affect both foals and adults, and they spread easily by horse-to-horse contact and by fomites. Seemingly well horses can harbor the bacteria, and when stressed, they can shed it or become ill. There are more than 2400 serotypes of Salmonella with S.typhimurium being more frequently isolated. These zoonotic agents cause commonly gastrointestinal disease and diarrhea, and rare abortion, septicemia; and in foals it can cause generalized sepsis. In adult horses, bacteria are more likely to be confined to the GI tract, particularly the colon. Horses of all age groups are susceptible. Multidrug resistant S. typhimurium DT104 (Salmonella enterica subspecies enterica Serotype Typhimurium Definitive Type 104) has significant zoonotic potential due to its high lethality rate in human beings as well. Initially S. typhimurium DT104 was isolated from cattle in 1988 in England and Wales but consequently was reported from sheep, pigs, poultry and horse. Fecaloral route with high inoculums size containing more number of bacteria is the most common way for zoonotic spread of salmonellosis in human population, though in immunocompromized persons low inoculums may also produce the disease. Higher mortality rate and resistance to commonly preferred antibiotics further aids to the potential of this pathogen for zoonotic transmission. Suspected cases should be monitored and treated separately. Strict follow up of personal hygienic measures and proper disinfection of stables, contaminated equipments and utensils will help in reducing the menace of zoonotic transmission (Fone and Barker, 1994; Weese et al., 2001; Weese, 2002).

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5.

Contagious equine metritis

Contagious equine metritis is caused by Taylorella equigenitalis (previously Haemophilus equigenitalis), which causes endometritis resulting in temporary infertility. It is a nonsystemic illness, the effects of which are restricted to the reproductive tract of the mare. The organism is Gram negative, oxidase positive, catalase positive and phosphatase positive. The chief clinical signs are a copious to slight mucopurulent vaginal discharge and a variable cervicitis and vaginitis. Recovery is uneventful, but prolonged asymptomatic carriage is established in a proportion of infected mares. The infection is most frequently transmitted by sexual contact with carrier stallions, which are always asymptomatic and in which the principal sites of T. equigenitalis colonisation are the urogenital membranes (urethral fossa, urethra and penile sheath). Inadequate hygiene during the cleansing or examination of the genitalia of horses can also be responsible for the transmission of infection. Carrier mares are colonised on the urogenital membranes, principally in the clitoral sinuses and fossa and very frequently in the uterus. Foals born of carrier mares may also become carriers. The organism can infect equid species other than horses, e.g. donkeys. Effective vaccines that protect against contagious equine metritis or prevent colonisation by T. equigenitalis are not yet available. Washing with disinfectants combined with local and systemic antibiotic treatment can eliminate T. equigenitalis. The principal means of control is through preventing transmission by the establishment of freedom from infection before breeding commences. Detection of infection depends on the culture of T. equigenitalis from urogenital swabs of mares and stallions and its accurate identification. Serum antibody to T. equigenitalis can be detected in mares for 3-7 weeks after infection and can also be demonstrated in the occasional carrier mare, but not stallion. Serology may therefore be of value in detecting recent, but not chronic, infection in the mare, but the emphasis for control of the disease should be on the detection of carriers by culture. 6.

Brucellosis

Brucellosis is one of the most important disease problems of animals and human beings. Almost all domestic species can be affected with brucellosis except cats which are resistant to Brucella infection. Considering the damage done by the infection in animals in terms of decreased milk production, abortions, weak offsprings, weight loss, infertility and lameness, it is one of the most serious diseases of livestock. It is also a major impediment for the trade. Death may occur as a result of acute metritis, followed by retained fetal membranes. Brucellosis is caused by members of genus Brucella. These are small, non-motile, aerobic, facultative intracellular, Gram-negative coccobacilli. The species of Brucella and their major hosts are Br. abortus (cattle), Br. Melitensis (goats), Br. suis (swine) and Br. ovis (sheep). This is caused by Brucella abortus in equines, and is manifested by fistulous withers, poll evil, lameness due to joint infection and rarely late abortions in mares.

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Horizontal transfer of Brucella spp. to horses from cattle and pigs has been documented (Forbes, 1990). Brucellosis is considered to be an occupational disease of slaughter house workers, butchers, animal handlers and veterinarians. This causes undulant fever in human beings. Transmission typically occurs through contact with infected animals or materials with skin abrasions. The incubation period in humans varies from 1-3 weeks. The symptoms may include irregular fever, headache, weakness, malaise, profuse sweating during night. Coughing, chest pain, irritation, insomnia, depression are occasionally encountered. During the early stage of the disease, patients are frequently bacterimic, making circulating Brucella detectable by blood culture. In many patients, the symptoms last for 2 to 4 weeks and are followed by spontaneous recovery. Others develop recurrent bouts at 2-14 day intervals. Most people with this undulant form recover completely in 3 to 12 months. A few patients become chronically ill, with symptoms of chronic fatigue, depressive episodes and arthritis. Occasional complications include arthritis, endocarditis, granulomatous hepatitis, meningitis, uveitis, orchitis, cholecystitis, osteomyelitis, and rare cases of encephalitis. Asymptomatic infections are also common in humans. Though it has been eradicated in many developed countries in Europe, Australia, Canada, Israel, Japan and New Zealand, yet it remains an uncontrolled problem in regions of high endemicity such as the Africa, Mediterranean, Middle East, parts of Asia and Latin America. Ehizibolo et al. (2011) have reported a seroprevalence of 14.7% of equine brucellosis in Nigeria. Tahamtan et al. (2010) have reported a seroprevence of 2.5% for brucellosis in horses in Iran. A seroprevalence of 5.88%, 12.89% and 5.78% has been reported from Egypt (Montasser et al., 1999), India (Sharma et al., 1979) and Pakistan (Ahmed and Munir, 1995a,b), respectively. Diagnosis conventionally relies on the detection of circulating antibodies followed by the bacteriological isolation and serum agglutination tests (Rose Bengal plate agglutination test (RBPT) and standard tube agglutination test (STAT). Molecular diagnostic assays can be used for detection instead of serological tests because serological assays like RBPT, STAT have the disadvantage of false positive reaction against other gram negative bacteria. Various molecular assays like polymerase chain reaction (PCR), Real time-PCR, etc., can be employed. Recent diagnostics like loop mediated isothermal amplification assay and lateral flow assay can be employed (Karthik et al., 2014a; Karthik et al., 2014b). A Light Cycler-based real-time PCR (LC-PCR) assay has been developed by Qeipo-ortuno (2005) to evaluate its diagnostic use for the detection of Brucella DNA in serum samples. Following amplification of a 223-bp gene sequence encoding an immunogenetic membrane protein (BCSP31) specific for the Brucella genus, melting curve and DNA sequencing analysis was performed to verify the specificity of the PCR products. The assay has a total processing time of 35.0% to 50.0 thousand serum adn milk samples of animals and human beings. Vaccination strategy Vaccination is the efficient and cost-effective strategy to prevention of the appearance of the clinical cases. ‘Indigenous Vaccine’ used in goats in India has helped to reduce the age of maturity and inter kidding period, improved per animal productivity and helped to conserve the threatened native breeds of domestic livestock due to JD in Indian conditions (Singh et al., 2011).Vaccination has been used as a tool to aid in the control of JD in many countries like (Australia, New Zealand, Spain, Netherlands, Denmark, Norway, Canada, Denmark, Iceland, France, etc., and now US is also adopting vaccine based control of Johne’s disease after failure of their much hyped ‘hygienic conditions’ and ‘test and cull’ methodology in controlling bovine JD by developing it’s own mutant vaccine. We at CIRG, Makhdoom have been engaged in maintaining the 3 goatherds of elite animals consist (nearly 2500) of 3 breeds endemic for Johne’s disease since establishment in 1976. To maintain the elite animals of 3 breeds endemic for Johne’s disease we employed ‘test and cull’ strategy to control JD for last 40 years but without success.

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Hence ‘test and cull’ strategy for the control of Johne’s disease at CIRG, Makhdoom, failed miserably, despite culling thousands of goats between 1976 and 2005. Therefore, we at CIRG in 2005, developed first ‘Indigenous Vaccine’ against Johne’s disease using highly pathogenic goat adopted novel MAP strain (S 5) of ‘Indian Bison Type’ biotype (Singh et al., 2007). Between 2006 and 2016, in ten years time >45 trials have taken place in the four domestic livestock species in different parts of the country. This goat based ‘Ingenious Vaccine’ has proved to be highly effective and animals (cattle, buffaloes, goats and sheep) suffering from clinical JD were cured of the disease, therefore, ‘Indigenous vaccine’ was both ‘therapeutic’ and preventive (Singh et al., 2007). The vaccine was given in the neck region below the skin subcutaneously in animals above 3 months of age. Though disease is endemic in native animals, but in some of the animals it produced swelling or ‘take’. In western countries, inoculation is given into the brisket area of calves less than one month old (preferably less than one week) producing considerable local reaction. Vaccinated herds/flocks get much reduced clinical cases and losses but JD will be eradicated remains question mark. However, vaccination may be the most cost-effective option for commercial herds breeding their own replacements and experiencing considerable loses from JD. A live vaccine is licensed for use in the UK, but can only be given to animals in the first month of life. This may result in a reduction of clinical disease in infected herds, but will not lead to eradication of infection. Vaccinated animals frequently break down when sold to other herds, negating the value of vaccination for herds selling breeding replacements. Vaccination has been reported to interfere with interpretation of the tuberculin skin test. There is also potential for interference with the skin test for tuberculosis. As a result of this and because of vaccination with a live organism that may be capable of causing disease in humans, live vaccines are not favoured by several countries. Killed vaccines are however used and positive cost benefit has been reported where ever used. Research into improved vaccines is being undertaken in many countries. Several studies have shown the profitability of vaccination for paratuberculosis (Juste and Casal, 1993; van Schaik et al., 1996). Over a period of a few years, economic advantages of vaccination may be up 20 times higher than test and cull strategy. Further it has been suggested that vaccination might be the beginning of the end of the huge worldwide problem of domestic livestock called ‘paratuberculosis’ and might mark the difference between doing nothing and advancing towards global control (Juste et al., 2002). Though there are several strategies for JD control, but there is no generalized consensus on which one or which combination of strategies should be the standard approach. This may be due to the fact that JD control programs emphasize too heavily MAP eradication. First ‘Indigenous vaccine’ developed by Singh et al. (2007), using novel, native, highly pathogenic, fully characterized and sequenced (Whole Genome sequencing) of ‘S 5’ strain of ‘Indian Bison Type’ biotype of MAP isolated from a clinically sick Jamunapari goat that died of JD at CIRG farm. Vaccine contained 2.5

47 

 

mg (dried weight) of heat inactivated native strain of MAP bacilli (approximately 12 x108cfu/ml) suspended in one ml of Aluminum hydroxide gel. This ‘Indigenous vaccine’ has been shown to provide good protection (prophylactic) in experimentally vaccinated and twice-challenged goats (Singh et al., 2007). There are a number of reports on the prophylactic properties of JD vaccine, whereas very few reports are available on the ‘therapeutic potential of JD vaccine globally (Srivastava et al., 2010, Singh et al., 2010). Large scale epidemiological studies by Singh et al. (2014) in domestic livestock species for past 28 (1985 to 2013) showed that JD disease is endemic and there is substantial increase in prevalence of disease in these 28 years. Molecular epidemiology showed that novel ‘Indian Bison Type’ MAP is the pre-dominant bio type infecting domestic and wild ruminant population (Sohal et al., 2009). Therefore all the 4 livestock species (goats, sheep, cattle and buffaloes) above 3 months of age were immunized with same ‘Indigenous JD vaccine’ initially developed and standardized for goats, with dose rate of 1 ml (goats and sheep)and 2 ml (cattle and buffaloes) of ‘Indigenous vaccine’ against MAP subcutaneously (behind the ear). Dose rate studies (Singh et al., 2014) at CIRG, during large scale ‘vaccine trials’ showed that internationally used dose was also optimum for our conditions. Any increase in dose led to stress on already weak animals. Since in Indian conditions the physical profile of animals is in general weak and stressed, due to improper nutrition and management of animals. However, our studies have shown that like incidence of diseases, the vaccine response was also dependant on the ‘status of nutrition’. Under optimum nutrition conditions one dose of vaccine was sufficient for life (Singh et al., 2013) but under deficient feeding conditions, re-vaccination of animals every year was required (Final report of CSIR-NMITLI project on JD vaccine, 2015). In our latest study (personal communication) on vaccination of goats under well fed conditions, in Jakhrana breed of goats (milch breed), exhibited that under the environmental conditions, where JD was endemic in goatherds and sharing of grazing land with non-vaccinated goats, the peak yield (3.85 liters of milk per day) was achieved in 3rd years after vaccination. However, at this point of time there was also slight shedding of MAP bacilli in feces, which showed that in well-fed conditions in goats, it was necessary to re-vaccinate the goats at 3rd year in conditions where disease was endemic in other goatherds sharing the grazing land. In conditions where disease is endemic due to repeated infection and lactation stress, animals will need re-vaccination at every 3 year interval. Vaccinated animals were monitored for 12 months (study period) at different time intervals for immune response by indigenous ELISA (Singh et al., 2009), shedding of MAP in faeces by microscopy (Singh et al., 2011) and changes in physical profiles and production parameters (Singh et al., 2013; 2015). Overall improvements on the basis of health (morbidity and mortality), production parameters (reproductive efficiency, milk production), physical and clinical conditions (weakness, diarrhea, skin coat) were measured. Author has the experience of using different adjuvants (Gerbu; mineral oil with liposomes, Germany, Aluminium hydroxide gel of Indian and CZ Veterinaria, Spain now taken over by Pfizer), Seppic, Montanide oil) but did not find any

48 

 

significant difference in efficacy and protection, except presentation and syringability. Some facts related to the recent researches on Johne’s disease vaccines have been specified below. An ideal JD vaccine should have following properties. i.

Ideal Johne’s disease Vaccine

a. b. c. d.

Cause minimal tissue injury No interference between tuberculosis and paratuberculosis diagnosis Discriminate between infected and vaccinated animals Eliminates fecal shedding of bacteria

Globally efforts are on to develop various type of vaccines with superior efficacy against incurable Johne’s disease of domestic livestock. ii.

Johne’s disease Vaccines under Developmentgobally

a. b. c.

Modified Live attenuated Whole Cell MAP Vaccines Killed whole cell MAP Vaccines Gene knockout whole live MAP vaccines; Live mutant vaccine: By random, direct and insertional mutagenesis. Killed cell wall deficient (CWD) whole cell Vaccines Vector based vaccine: Use of M.bovis BCG as vector to express MAP proteins Protein subunit vaccine MAP: Recombinant MAP Hsp70, 74F, Ag85AA, g85BAg, 85C, SOD. DNA vaccine: Plasmids DNA encoding cocktail of MAP Proteins

d. e. f. g.

In the global movement against Johne’s disease, USA (cattle or bovine JD) besides Canada (Ovine JD) is latest to join the bandwagon after the failure of ‘Test and cull’ methodology. However, efforts are mainly to develop a mutant vaccine and following are some of the examples of mutant vaccine under different stages of development. Table 1: Transposon mutant vaccine candidates of MAP Institutiona

Location of insertionb

References

MAP3006c (lipN)

MAP strainc Goat strain 43432-02 K-10

USDA-ARS-WRRC

MAP0482

University of Wisconsin Washington State University University of Nebraska University of Nebraska University of Nebraska University of Nebraska University of Nebraska University of Nebraska University of Nebraska New York, USA

MAP1047 (relA)

K-10

Park et al., 2014

MAP1566 MAP3695 and fadE5 MAP0460 (lsr2) MAP0282c and 0283c MAP1566 MAP2296c and 2297c MAP1150c and 1151c leuD

K-10 K-10 K-10 K-10 K-10 K-10 K-10 Strain 66115-98

University of Wisconsin

MAP1872c (mbtH_2)

ATCC19698

AgResearch NZ

MAP1566

strain 989

Rathnaiah et al., 2014 Rathnaiah et al., 2014 Rathnaiah et al., 2014 Rathnaiah et al., 2014 Rathnaiah et al., 2014 Rathnaiah et al., 2014 Rathnaiah et al., 2014 Faisal et al., 2013 Kabara and Coussens, 2012 Scandurra et al., 2010

49 

McGarvey, unpublished Bannantine, 2014

 

AgResearch NZ MAP0011 (ppiA) K-10 Scandurra et al., 2010 Washington State MAP3893c (pknG) K-10 Park et al., 2008a University Washington State MAP0460 (lsr2) K-10 Park et al., 2008a University University of Wisconsin MAP3963 (umaA1) ATCC19698 Shin et al., 2006 University of Wisconsin MAP2408c (fabG2_2) ATCC19698 Shin et al., 2006 a The location of the laboratory where the mutant(s) was constructed. b The MAP locus where the transposon inserted. If two genes are listed, the transposon is inserted in the intergenic region between the two. If the gene has been named, it is shown in parenthesis. c The parental strain of MAP used to create the mutation.

Internationally following vaccines have been widely used in different countries for the control of JD for period of 5 to 35 years and have shown remarkable reduction in the reduction of National prevalence of JD. Table 2: Johne’s disease vaccines in the International Market Sn

Name/ Kind of Vaccine Fromm Lio-Johne#

Vaccine strain and Adjuvant Countries type 1. MAP Strain 18, Killed Oil type (Freund’s complete) USA 2. 316F strain, Live Oil type Spain attenuated 3. Phylaxia 5889 Bergey, Killed Oil type Hungary 4. Weybridge 316F strain, Live Paraffin & Olive oil with United Vaccine attenuated Pumice stone powder Kingdom 5. Gudair# (Pfizer MAP Strain 318F, Oil emulsion Australia CSL) Killed 6. Aqua Vax Map Strain 316F, Live Water based (saline) New attenuated Zealand 7. Neoparasec Freeze Dried Live Oil type France (MerialNZ Ltd.) MAP, Live attenuated 8. Mycopar# Whole cell bacterin, Oil emulsion Germany inactivated 9. Silirum (Pfizer MAP Strain 318F, Oil emulsion Australia CSL) Killed 10. Bio-JD Oil & Gel Native MAP strain ‘S Aluminium hydroxide gel India (2004 (Biovet Pvt. 5’ Indian Bison type, (Alum) and Seppic oil to 2014)* Ltd.)* Killed # For sheep and goats, *For goats, sheep, cattle and buffaloes and has been licensed by Drug Controller, Government of India (DCGI, New Delhi) and ‘Candidate vaccine strain and technology has been transferred to M/S BIovetpvt.ltd. Table 3: Countries where vaccination experiments* carried out (Bastida and Juste, 2011). Percent (Of total trials Percent (Of total trials Sn Countries Experiments n upto 2010)# upto 2014) 1 Australia 12 10.2 8.2 2 Denmark 1 0.8 0.6 3 France 5 4.2 3.4 4 Germany 1 0.8 0.6 5 Greece 6 5.1 4.1

50 

 

6 7 8 9 10 11 12 13 14

Hungary 1 0.8 0.6 Iceland 2 1.7 1.3 India 4, now 32 3.4 21.9 Netherlands 12 10.2 8.2 New Zealand 17 14.4 11.6 Norway 1 0.8 0.6 Spain 16 13.6 10.9 United Kingdom 9 7.6 6.1 United States 31 26.3 21.2 Total 118, now 146 100 100 *An experiment is defined as vaccine trial whose results are measured according to one of the three outcome variables: clinical signs, MAP isolation, gross or microscopic lesions. InIndia, total 32 vaccine trials/studies were completed. #Bastida and Juste Journal of Immune Based Therapies and Vaccines 2011, 9:8 doi:10.1186/14768518-9-8

Monitoring parameters for ‘therapeutic vaccine trials’ a. Herd profile: Age (6-12 months, 12-18 months & adult) and sex-wise (males/females) profile of the animals in the herds would be prepared. All the animals in a herd above 3 months of age will be vaccinated with ‘Indigenous vaccine’ as above irrespective of physiological stage and health (clinical condition (sub-clinical, clinical and advance clinical) with respect to MAP infection. b. History of Johne’s disease: On the basis of history, mortality, morbidity, necropsy, screen of the farms, cullings for Johne’s disease infection, status of Johne’s disease would be estimated. c. Screening of animals before vaccination: Faecal, serum, blood and milk samples of 25.0% animals will be screened twice at monthly intervals using microscopy, culture, ELISA kit and PCR. All the animals above 3 months of age will be vaccinated irrespective of sex and physical and physiological condition of the animals. d. Monitoring of Vaccinated animals: The vaccinated and control groups will be monitored on following parameters from zero day and after vaccination upto 360 DPVon the basis of health (mortality, morbidity, etc.) body condition score and production (birth weights, body weights, reproductive efficiency etc.), physical condition (diarrhea, weakness, etc), immunological parameters (ELISA titer) and status of shedding of MAP. However, necropsy findings- animals died during experiments has been examined for presence and absence of gross and microscopic lesions of JD in visceral organs and particularly in mesenteric lymph nodes and intestinal mucosa whereas improvements in growth rates, gain and loss in body weights would be measured at monthly intervals. 1.

Effects of vaccination on shedding of MAP bacilli Screening of fecal samples at different time points (days post vaccination) showed that there was marked reduction (5.8-99.1%) in shedding of MAP bacilli in feces (Table 3).Studies showed vaccination is the best strategy to control JD, because it yielded approximately 3 to 4 times better benefit-to-cost ratios than other 51 

 

strategies (Juste and Casal, 1993). Vaccination improves the immunity of individuals are able to arrests the progression of the infection results in reduction of the shedding of bacilli. Table 4: Effects of vaccination of shedding of MAP bacilli Sn

Name / Country Species Kind of Vaccine 1. Laboratory USA Cattle Scale (Live) 2. Fromm USA Cattle (Killed) 3. Live USA Cattle attenuated 4. Laboratory Denmark Cattle Scale (Live) 5. Laboratory France Cattle Scale (Live) 6. Phylaxia Hungary Cattle (Killed) 7. Neoparasec Germany Cattle (Live) 8. Lio-Johne Spain Sheep (Live) 9. Laboratory Greece Sheep Scale (Live) 10. Gudair Australia Sheep (Gudair) 11. Laboratory India Goat Scale (Killed) * 12. Laboratory India Goat Scale (Killed) * 13. Laboratory India Sheep Scale (Killed) * 14. Laboratory India Sheep Scale (Killed) * 15. Laboratory India Goat Scale (Killed) * 16. Laboratory India Cattle Scale (Killed) * 17. Laboratory India Cattle Scale (Killed) * * Indigenous vaccine now commercialized

Reduction (%)

Reference

81.4

Period of study -

99.1

-

Hurley et al., 1983

90.0

-

92.9

-

Saxegaard & Fodstad, 1985 Jorgensen, 1983

81.6

5 years

Argente, 1992

94.7

-

Kormendy, 1994

86.8

-

Klawonn et al., 2002

80.8

-

Aduriz, 1993

93.2

-

90.0

-

Dimareli-Malli et al., 1997 Eppleston et al., 2004

82.1

-

Singh et al., 2007

62.1

7 months

Singh et al., 2010

27.3

3 years

Singh et al., 2013a

17.1

4 months

Shroff et al., 2013

5.8

4 months

Singh et al., 2013b

46.6

6 months

Rawat et al., 2014

89.3

9 months

Singh et al., 2015

52 

Larsen et al., 1974

 

2.

Effects of vaccination on production

Vaccine programs strongly suggested that vaccination has more a therapeutic than a preventive effect, as confirmed by the positive results obtained when vaccinating adults (Juste and Perez, 2011). Indian vaccination trials also confirmed the therapeutic effects of vaccination, however, these did not focused on difference in preventive and therapeutic values (Singh et al., 2010c; Singh et al., 2011). Vaccination prevents clinical cases and thus may lead to increased production at a highly profitable benefit-to-cost ratio (Table 4). Table 5: Effects of vaccination on production (mortality or clinical cases) Sn 1. 2.

Name / Kind of Vaccine Weybridge (Live) Lelystad (Killed)

Country UK Netherlands

3. 4.

Lio-Johne (Live) Gudair (Killed)

5.

Neoparasec (Live)

Cattle Cattle

Reduction (%) 99.06 91.82

Period of study -

Spain Australia

Sheep Sheep

78.29 87.5

-

New Zealand

Sheep

71.43

One year

Goat

82.78

-

Goat

54.8

7 months

Goat

40.0

4 months

Cattle

95.0

6 months

6.

Species

Laboratory Scale Greece (Live) 7. Laboratory Scale India (Killed)* 8. Laboratory Scale India (Killed) * 9. Laboratory Scale India (Killed) * * Indigenous vaccine now commercialized

3.

Reference Wilesmith, 1982 Kalis et al., 1992 Aduriz, 1993 Windsor et al., 2003 Gwozdz et al., 2000 Xenos et al., 1988 Singh et al., 2010 Singh et al., 2013b Rawat et al., 2014

Effects of vaccination on histological lesions

Vaccination modifies the immune-pathologic processes that lead to the persistent progressive regional intestinal inflammation responsible for clinical disease in such a way that immunized individuals are able to arrest the progression of the infection and the ensuing lesions. This results in reduction of the excretion of MAP and significant decrease in the severity of clinical signs and economic losses (Table 5). According to a 1985 report, vaccination resulted in a 98.0% reduction in postmortem finding of lesions, which, during a period of 16 years, reduced incidence from 53.0% to 1.0% (Saxegaard and Fodstad, 1985). Table 6: Effects of vaccination on histological lesions Sn 1 2

Name / Kind of Vaccine Laboratory Scale (Killed) Silirum (Killed)

Country The Netherlands Spain

Species Cattle

Reduction (%) 58.9

Period of study 12 years

Cattle

38.6

-

53 

Reference van Schaik et al., 1996 García-Pariente et al., 2005

 

3

Iceland

Sheep

93.5

-

Sigurdsson,

4 5

Laboratory Scale (Killed) Lio-Johne (Live) Mycopar (Killed)

Spain USA

Sheep Sheep

100.0 75.3

-

6

Gudair (Killed)

Australia

Sheep

72.7

5 years

7

Gudair (Killed)

New Zealand Norway

Sheep

75.5

Laboratory Scale Goat (Live) 9 Gudair (Killed) Spain Goat 10 Laboratory Scale USA Goat (Killed) 11 Laboratory Scale India Goat (Killed)* 12 Laboratory Scale India Goat (Killed) * 13 Laboratory Scale India Cattle (Killed) * * Indigenous vaccine now commercialized

97.1

16 months 14 years

Aduriz, 1993 Thonney and Smith, 2005 Reddacliff et al., 2006 Griffin et al., 2009

8

65.8 66.6 75.0 57.1 66.7

~9 months 7 months 4months

Saxegaard and Fodstad, 1985 Corpa et al., 2000 Kathaperumal et al., 2009 Singh et al., 2007 Singh et al., 2010 Singh et al., 2015

The indigenous vaccine developed at CIRG, has been extensively tried in the 4 species of domestic livestock in different locations and management conditions over period of past 10 years and has shown excellent performance and vaccination of advance cases of JD animals have come back in to the production and health. References Aduriz JJ (1993). Epidemiologia, diagnóstico y control de la paratuberculosisovina en la ComunidadAutónomadel País Vasco. University of Zaragoza, Spain. Argente G (1992). Efficiency of vaccination and other control measures estimated by fecal culturing in a regional program. In Proceedings of the 3rd International Colloquium on Paratuberculosis; Orlando, Florida, USA. Edited by Chiodini RJ, Kreegel JM. International Association for Paratuberculosis, pp. 495-503. Ayele WY, Machackova M, Pavlik I. (2001). The transmission and impact of paratuberculosis infection in domestic and wild ruminants. Vet. Med. Czech. 46(7-8): 205-224. Bastida Felix and Juste RA (2011). Paratuberculosis control: a review with a focus on vaccination. Journal of Immune Based Therapies and Vaccines. 9: 8. Corpa JM, Peerez V & Garcia Marin JF, Differences in the immune responses in lambs and kids vaccinated against paratuberculosis, according to the age of vaccination, Vet Microbiol, 77 (2000) 475. Dimareli-Malli Z, Sarris K, Papadopoulos O, N I, Xenos G, A M, Papadopoulos G (1997). Evaluation of an inactivated whole cell experimental vaccine against paratuberculosis in sheep and goats. PTBC Newsletter, 9: 10-17. Eppleston J, Reddacliff L, Windsor P, Whittington R, Jonbes S (2004). Field studies on vaccination for the control of OJD in Australia-and overview.ProcAust Sheep Vet Soc., 5659. FAO (2013). FAOSTAT. Food and Agriculture Organization, Rome.

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Garcia-Pariente C, Pérez V, Geijo M, Moreno O, Muñoz M, Fuertes M, Puentes E, Doce J, Ferreras MC, Garcia Marin JF. The efficacy of a killed vaccine against paratuberculosis (SILIRUM®) in cattle. A field study. In Proceedings of the 8th International Colloquium on Paratuberculosis; Copenhagen, Denmark. Edited by Manning EJB, Nielsen SS. International Association for Paratuberculosis; 2005: 52. Griffin JF, Hughes AD, Liggett S, Farquhar PA, Mackintosh CG, Bakker D (2009). Efficacy of novel lipid-formulated whole bacterial cell vaccines against Mycobacterium avium subsp. paratuberculosis in sheep. Vaccine. 27: 911-918. Gwozdz JM, Thompson KG, Manktelow BW, Murray A, West DM (2000). Vaccination against paratuberculosis of lambs already infected experimentally with Mycobacterium avium subspecies paratuberculosis. Aust Vet J., 78: 560-566. Hurley S, Ewing E. Results of a field evaluation of a whole cell bacterin. In Proceedings of the International Colloquium on Paratuberculosis, I; NADC, USDA, Ames, IA, USA. Edited by Merkal RS. International Association for Paratuberculosis; 1983: 244-248 Jorgensen JB (1983). The effect of vaccination on the excretion of Mycobacterium paratuberculosis. In Proceedings of the International Colloquium on Paratuberculosis, I; NADC, USDA, Ames IA, USA. Edited by Merkal RS. International Association for Paratuberculosis, pp. 249-254. Juste RA and Casal J (1993). An economic and epidemiologic simulation of different control strategies for ovine paratuberculosis. Prev. Vet. Med. 15: 101-115. Juste RA, Geijo MV, Sevilla I, Aduriz G and Garrido JM (2002). Control of paratuberculosis by vaccination. In Proceedings of the 7th International Colloquium on Paratuberculosis; Bilbao, Spain. Edited by: Juste RA. International Association for Paratuberculosis. pp. 331. Kalis CH, Hesselink JW, Barkema HW, Collins MT (2001). Use of long-term vaccination with a killed vaccine to prevent fecal shedding of Mycobacterium aviumsubspparatuberculosisin dairy herds. Am. J. Vet. Res. 62(2): 270-274. Kalis CHJ, Benedictus G, van Weering HJ, Flamand F, Haagsma J (1992). Experiences with the use of an experimental vaccine in the control of paratuberculosis in The Netherlands. In Proceedings of the 3rd International Colloquium on Paratuberculosis; Providence, RI, USA. Edited by Chiodini RJ, Kreeger JM. International Association for Paratuberculosis, pp. 484494. Kathaperumal K, Kumanan V, McDonough S, Chen LH, Park SU, Moreira MA, Akey B, Huntley J, Chang CF, Chang YF (2009). Evaluation of immune responses and protective efficacy in a goat model following immunization with a coctail of recombinant antigens and a polyprotein of Mycobacterium avium subsp. paratuberculosis. Vaccine. 27:123-135. Klawonn W, Cussler K, Drager KG, Gyra H, Kohler H, Zimmer K, Hess RG (2002). The importance of allergic skin test with Johnin, antibody ELISA, cultural fecal test as well as vaccination for the sanitation of three chronically paratuberculosis-infected dairy herds in Rhineland-Palatinate. DtschTierarztlWochenschr., 109: 510-516. Kormendy B (1994). The effect of vaccination on the prevalence of paratuberculosis in large dairy herds. Vet Microbiol., 41: 117-125. Larsen AB, Merkal RS, Moon HW (1974). Evaluation of a paratuberculosis vaccine given to calves before infection. Am J Vet Res., 35: 367-369 Rawat KD, Chaudhary S, Gupta S, Chaubey KK, Jayaraman S, Kumar N, Sohal JS, Sachan TK, Dhama K, Singh SV (2014). Potential of ‘goat based vaccine’ using ‘India bison biotype’ of Mycobacterium aviumsubspecies paratuberculosisin salvaging a dairy farm consisting of high yielding Holstein Fresian cows from devastation and closure due to outbreak of bovine Johne’s disease in Northern India. Adv. Anim. Vet. Sci. 2 (12): 638-646.

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Reddacliff L, Eppleston J, Windsor P, Whittington R, Jones S (2006). Efficacy of a killed vaccine for the control of paratuberculosis in Australian sheep flocks. Vet Microbiol., 115:77-90. Saxegaard F, Fodstad FH (1985). Control of paratuberculosis (Johne’s disease) in goats by vaccination. Vet Rec., 116: 439-441. Shroff S, Chandel BS, Dadawala AI, Singh SV, Bhagat AG, Chauhan HC & Gupta S, Evaluation of Indigenous vaccine in Patanwadi sheep naturally infected with clinical Johne’s disease, Res OpinAnim Vet Sci, 3 (2013) 322. Sigurdsson B (1960). A killed vaccine against paratuberculosis (Johne’s disease) in sheep. Am J Vet Res., 21: 54-67. Singh AV, Singh SV, Sohal JS, and Singh PK (2009). Comparative potential of modified indigenous, Indigenous and commercial ELISA kits for diagnosis of Mycobacterium avium subspecies paratuberculosis in goat and sheep. Indian J. Exp. Biol. 47(5): 379-382. Singh K, Chandel BS, Chauhan HC, Dadawala A, Singh SV & Singh PK (2013). Efficacy of ‘Indigenous Vaccine’ using native ‘Indian Bison Type’ genotype of Mycobacterium aviumsubspecies paratuberculosis for the control of clinical Johne’s disease in an organized goat herd, Vet Res Commun, 37: 109. Singh SV, Singh AV, Singh PK, Gupta S, Singh H, Singh B, VinodhKumar OR, Rajendiran AS, Swain N &Sohal JS (2013a). Evaluation of ‘Indigenous vaccine’ developed using ‘Indian Bison Type’ genotype of Mycobacterium aviumsubspecies paratuberculosis strain ‘S5’ of goat origin in a sheep flock endemic for Johne’s disease: A three years trial in India, World J Vaccines, 3: 52. Singh SV, Singh PK, Singh AV, Sohal JS, Kumar N, Chaubey KK, Gupta S, Rawat K D, Kumar A, Bhatia A K, Srivastav A K, Dhama K (2014a). Bio-load and bio-type profiles of Mycobacterium aviumsubspeciesparatuberculosis infection in the farm and farmer’s herds/ flocks of domestic livestock: A 28 years study (1985-2013). Transbound. Emerg. Dis. 61 (Suppl. 1): 43-55. Singh SV, Singh PK, Singh AV, Sohal JS, Sharma MC (2010). Therapeutic effects of a new ‘Indigenous Vaccine’ developed using novel native ‘Indian Bison type’ genotype of Mycobacterium avium subspecies paratuberculosisfor the control of clinical Johne’s disease in naturally infected goatherds in India. Vet. Med. Int. doi:10.4061/2010/351846 Singh SV, Solanki M, Kumar A, Singh P K, Singh AV, Singh B &Sohal JS (2011). Comparative evaluation of improved ‘Modified Microscopic Test’ with traditional microscopy, indigenous ELISA kit, fecal and blood PCR for the diagnosis of Mycobacterium aviumsubspecies paratuberculosis in goatherd endemic for Johne’s disease, Research & Reviews: A Journal of Life Sciences, 1: 8. Singh SV, Gupta S, Singh PK, Sohal JS, Kumar N, Kumar A,Chaubey KK, Singh B. 2013. Prophylactic study of ‘Indigenous vaccine’ against Johne’s disease in dairy cow and male calf of Hariana breed: case study. AdvAnim Vet Sci.1:23-28. Singh SV, Singh PK, Kumar N, Gupta S, Chaubey KK, Singh B, Srivastav A, Yadav S, Dhama K (2015). Evaluation of goat based ‘indigenous vaccine’ against bovine Johne’s disease in endemically infected native cattle herds. Indian J Exp Biol., 53(1): 16-24. Singh, S.V., Singh, P.K., Singh, A.V., Sohal, J.S., Gupta, V.K. and Vihan, V.S. (2007). Comparative efficacy of an indigenous ‘inactivated vaccine’ using highly pathogenic field strain of Mycobacterium avium subspecies paratuberculosis ‘Bison type’ with a commercial vaccine for the control of Capri-paratuberculosis in India. Vaccine, 25: 7102-7110. Sohal JS, Sheoran N, Narayansamy K, Brahmachari V, Singh SV, Subhod S. (2009). Genomic analysis of local isolate of Mycobacterium aviumsubspecies paratuberculosis. Vet microbial. Doi:10.1016 / J.Vetmic. 2008.08.027

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Srivastava, A.K. (2010). Prophylactic and therapeutic effect of Johne’s disease vaccine in cattle Pandit Deen Dayal Upadhyaya Pashu Chikitsa Vigya Vishwavidyalya Evam Go Anusandhan Sansthan, Mathura, Uttar Pradesh. Thonney ML SS, Smith MC. Control of Johne’s disease in sheep by vaccination Preliminary Report. In Control of Johne’s disease in sheep by vaccination Preliminary Report. Cornell University; 2005. vanSchaik G, Kalis CH, Benedictus G, Dijkhuizen AA and Huirne RB (1996). Cost-benefit analysis of vaccination against paratuberculosis in dairy cattle. Vet. Rec. 139: 624-627. Verma DK (2013). Mycobacterium avium subspecies paratuberculosis: an Emerging Animal Pathogen of Global Concern. Adv. Biores. 4(4): 01-08. Wilesmith JW (1982). Johne’s disease: a retrospective study of vaccinated herds in Great Britain. Br Vet J., 138: 321-331. Windsor PA, Eppleston J, Sergeant E (2003). Monitoring the efficacy of Gudair™ OJD vaccine in Australia. ProcAust Sheep Vet Soc., 114-122. Xenos G, Yiannati A, Dimarelli Z, Mtliangas P, Koutsoukou E: Evaluation of a live paratuberculosis vaccine in sheep and goats. In CEC Workshop Commission of the Economic Communities; Crete, Greece Edited by PO. 1988.

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Role of inflammation in severity of viral disease with focus on Herpes Papilloma virus Maryam Dadar and Kuldeep Dhama1 Razi Vaccine and Serum Research Institute, Agricultural Research, Education and Extension Organization (AREEO), Karaj, Iran 1 Division of Pathology, ICAR-Indian Veterinary Research Institute, Izatnagar-243122 (UP) India

Introduction Viral disease are among the numerous agents that can induce inflammatory pathways (Boccardo et al., 2010). The role of inflammation in viral infection is complicated since it plays vital role in controlling of initial infections, persistence of viral disease and development of related lesions (Mangino et al., 2015). Inflammatory responses is the most important lines of defense against virus infection. These responses are rapidly motivated to inhibit the viral spread during the initial hours after the infection, when the host may not yet be ready to reveal potent humoral and cellular immune responses against the invading virus (Bhat et al., 2011). Avoiding the immune response has been explored as a significant aspect of virus persistence which is the key factor leading to viral associated neoplasia. This finding proposes that the capability for limitation of host inflammatory responses would be worthwhile to the virus. Therefore, such limitations might be challenging to achieve, due to the numerous effecting mediators pathways for these processes. Also, viruses may break peripheral self-tolerance, and induce chronic inflammation (Rosa et al., 1998). Moreover, association between chronic inflammation and cancer in several organs support by clinical, epidemiologic, and animal studies (Lin and Karin, 2007; Mantovani et al., 2008). Between different viruses, the role of inflammation in human papillomavirus (HPV) remains poorly understood. Moreover, host inflammatory process may induce progression of lesion and affect tumor development by diverse mechanisms including the direct participation of inflammatory cells. In this review, we depict the interaction between HPV and inflammatory processes that finally may lead to cancer. Herpes Papilloma virus as a disease HPVs are a big family of non-enveloped, small, circular dsDNA viruses which surrounded by an icosahedral protein capsid predominantly comprising highly immunogenic L1 protein with a minor contribution from L2 protein (Leggatt et al., 2007, Frazer et al., 2004). This virus is the cause of proliferations of squamous epithelial cells or common warts on areas of the body such as the hands, feet anus, cervix, scrotum, groin, thigh, or penis (Stanley et al., 2012). HPV is reported as the main cause of cervical cancer in laboratory and epidemiological investigations (Castle et al., 2001). Also, recent data reveals that DNA of HPV can be detected in the most of cervical cancers.

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Moreover, HPV can develop cervical and other cancers such as cancer of the vulva, vagina, penis, or anus. It can also cause cancer in the back of the throat, including the base of the tongue and tonsils (Gillison et al., 2014). In a study demonstrated that inflammation of cervix may be connected with high-grade lesions and could be a cofactor for high-grade lesions of cervix in women infected with oncogenic HPV (Castle et al., 2012). Also, it is revealed that chronic local inflammation by development oral HPV infection or independently could be have an important role in the etiology head and neck squamous cell carcinoma (Tezal et al., 2012). Herpes Papilloma virus and its replication HPV invade to keratinocytes of the basal layers in the epithelial cell at a variety of anatomical sites and their replicative cycle is intimately linked with the differentiation of the infected cell (Mantovani et al., 2001). Amplification of HPV genome, expression of late gene and virion mounting occur in differentiated squamous epithelial cells that have recluse from the cell cycle (Boccardo et al., 2010). The high-risk types of mucosal HPV include as HPV-16 and HPV-18, induce lesions that can lead to cervical carcinoma, as well as express two proteins, E6 and E7, with proved oncogenic potential. In contrast, HPV-6 and HPV-11, are well characterized as the low-risk types which induce non-malignant genital warts and are very hardly associated with malignancies (zurHausen and Schneider, 1987). During HPV infection E6 involves in interfering with several cellular pathways in order to create a supportive environment for viral replication (Mantovani et al., 2001). Also, protein E7 of HPV-16 involve during the growth of cervical cancer by immunoinhibition and proliferation of tumor cell via the TGF-h1/Smads signaling pathway (Xu et al., 2006). Cellular responses to Herpes Papilloma virus infection The expression of some cytokines such as anti-inflammatory cytokine of IL-10 was studied in both normal and abnormal cervix; it was revealed that the squamous intraepithelial lesions severity in HPV infection is associated with the elevated expression of IL-10 (Shekari et al., 2012). High level of this immunomodulatory cytokine was explored to be localized to the transformation zone (Clerici et al., 1998). A study revealed that North Indian passive smokers having cytokines genotype of Rp1/Rp2 of IL-4 and genotypes of AC of IL-10 with anti-inflammatory properties had an increased risk for developing cervical cancer (Shekari et al., 2012). Also, it is suggested that Th2 cytokine genes may have a vital role in developing cervical cancer. Molecular studies have reported that inflammatory cytokines such as interleukin-1 (IL-1), IL-6, and TNFα, regulate proliferation of HPV and expression of its oncogenes E6 and E7 in cervical epithelial cells (McKaig et al., 1998, Iglesias et al., 1995; Galotti et al., 2000). Furthermore, a nonsteroidal anti-inflammatory drug has been explored to motivate HPV oncoproteins degradation, resulting to inhibition of growth and apoptosis of cervical carcinoma cells (Castle et al., 2003, Karl et al., 2007). In the other hand, both E6 and E7 oncoproteins of human

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papillomavirus 16 limit production of IL-18-induced IFN-γ in human peripheral blood mononuclear and NK cells (Lee et al., 2001). NF-κB expression is up regulated by E6 and E7 proteins of HPV16 and may help conserve against cervical cancer (Textor et al., 2010). HPV is also capable to regulate the tumor microenvironment through the production and release of specific pro-inflammatory cytokines and chemokines possibly to interact with the trafficking of leucocytes and/or allowing a better tumor development and infiltration (Lam et al., 2016). Activation of transcription factors The transcription factor AP-1 involves in the transcriptional modulation of specific types of high-risk human papillomaviruses (HPVs) such as HPV16 and HPV18, which are etiologically linked with the cancer development of the uterine cervix in women (Prusty et al., 2005). It is determined that the early transcription factors NF-κB, AP-1, and NF-IL6 are constitutively stimulated in human head and neck squamous cell carcinomas(HNSCC)cell lines and that NF-κB and AP-1 increased expression of the pro-inflammatory and pro-angiogenic cytokine IL-8 in HNSCC (Ondrey et al., 1999). Cytokine interplay in innate and adaptive immunity Recent epidemiologic evidence suggests some HPV cofactors, including nutritional factors, cervical inflammation, and genital tract infections (e.g., Chlamydia trachomatis and herpes simplex virus type 2 [HSV-2]) to be directly connected to the physiologic and immunologic state of the cervix, such as the cervical microenvironment (Castle et al., 2003). Interestingly, a switch from Thelper lymphocyte type 1 responses (Th1) (cell-mediated immunity) to T-helper lymphocyte type 2 (Th2) (humoral immunity) is revealed to happen during chronic inflammation (O’Byrne et al., 2001). It is well known that responses of Th1 are critical for response of host to intracellular pathogens and infectious diseases, and it is proposed that Th1 responses may have important role in the deletion of HPV infections (Scott et al., 2001). A vast switch from Th1 to Th2 cytokine production has also been connected with considerable HPV infection (Clerici et al., 1997). One study reported that in vitro interleukin 2 levels, a marker for Th1 responses to HPV antigens, to be negatively connected with status of disease (Tsukui et al., 1996). The high-risk HPV E6 and E7 oncoproteins also involves in the deregulation of innate immunity by interfering with the expression of Toll-like receptors (Kawai et al., 2009). Oncogenic HPV and inflammation HPVs have revealed various ways of targeting immune signaling pathways. Moreover, host inflammatory process may promote progression of lesion and affect development of tumor by diverse mechanisms including the direct participation of inflammatory cells (Lam et al., 2016). A lot of literature proposed that cervical carcinogenesis is also associated with inflammation (Boccardo et al., 2010; Shekari et al., 2012). High rates of cervical cancer often coincide with epidemic and endemic cervicitis as well as high-grade cervical lesions in the oncogenic HPV infected

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women (Castle et al., 2001). Another study demonstrated elevated expression of COX-2 in human cervical cancer, postulating that inflammation is connected to cervical carcinogenesis (Kulkarni et al., 2001). Also, epidemiologic studies report that chronic inflammation of the cervix induces the risk for cervical HPV infection and cervical cancer. In addition, the oral cavity and cervix are lined with similar types of mucosa, and the same HPV types cause cervical cancer and head and neck squamous cell carcinoma (HNSCC). Conclusion It is important to worry about severe inflammation, and pathways the HPVs overcome the significant barriers of the skin immune system. In this short review, we discuss some of interaction between HPV oncogenic proteins and inflammatory responses that finally may induce cancer. In the other word, HPV is able to regulate the tumor microenvironment through the production and release of specific proinflammatory cytokines and chemokines. Although the molecular mechanisms that regulate selective recruitment of inflammatory cells to the oral cavity and cervix and their activation following HPV infection are still poorly known. References Bhat, P., Mattarollo, S.R., Gosmann, C., Frazer, I.H. and Leggatt, G.R., 2011. Regulation of immune responses to HPV infection and during HPV‐directed immunotherapy. Immunological reviews, 239(1), 85-98. Boccardo, E., Lepique, A.P. and Villa, L.L., 2010. The role of inflammation in HPV carcinogenesis. Carcinogenesis, 31(11), 1905-1912. Castle, P.E., Hillier, S.L., Rabe, L.K., Hildesheim, A., Herrero, R., Bratti, M.C., Sherman, M.E., Burk, R.D., Rodriguez, A.C., Alfaro, M. and Hutchinson, M.L., 2001. An association of cervical inflammation with high-grade cervical neoplasia in women infected with oncogenic human papillomavirus (HPV). Cancer Epidemiology Biomarkers & Prevention, 10(10), 1021-1027. Castle, P.E. and Giuliano, A.R., 2003. Genital tract infections, cervical inflammation, and antioxidant nutrients-assessing their roles as human papillomavirus cofactors. National Cancer Institute monograph journal. 31, 29-34. Clerici M, Searer GM, Clerici E. Cytokine dysregulation in invasive cervical carcinomas and other human neoplasias: time to consider the Th1/Th2 pardigm. Journal of the National Cancer Institute. 1998;90: 261-266. Clerici, M., Ferrario, E., Trabattoni, D., Villa, M.L., Clerici, E., Merola, M., Stefanon, B., De Palo, G., Venzon, D.J. and Shearer, G.M., 1997. Cytokine production patterns in cervical intraepithelial neoplasia: association with human papillomavirus infection. Journal of the National Cancer Institute, 89(3), 245-250. Frazer, I.H., 2004. Prevention of cervical cancer through papillomavirus vaccination. Nature Reviews Immunology, 4(1), 46-55. Iglesias, M., Plowman, G.D. and Woodworth, C.D., 1995. Interleukin-6 and interleukin-6 soluble receptor regulate proliferation of normal, human papillomavirus-immortalized, and carcinoma-derived cervical cells in vitro. The American journal of pathology, 146(4), p.944. Gaiotti, D., Chung, J., Iglesias, M., Nees, M., Baker, P.D., Evans, C.H. and Woodworth, C.D., 2000. Tumor necrosis factor‐α promotes human papillomavirus (HPV) E6/E7 RNA

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expression and cyclin‐dependent kinase activity in HPV‐immortalized keratinocytes by a ras‐dependent pathway. Molecular carcinogenesis, 27(2), 97-109. Gillison, M.L., Castellsagué, X., Chaturvedi, A., Goodman, M.T., Snijders, P., Tommasino, M., Arbyn, M. and Franceschi, S., 2014. Eurogin Roadmap: comparative epidemiology of HPV infection and associated cancers of the head and neck and cervix. International journal of cancer, 134(3), 497-507. Karl, T., Seibert, N., Stöhr, M., Osswald, H., Rösl, F. and Finzer, P., 2007. Sulindac induces specific degradation of the HPV oncoprotein E7 and causes growth arrest and apoptosis in cervical carcinoma cells. Cancer letters, 245(1), 103-111. Kawai, T. and Akira, S., 2009. The roles of TLRs, RLRs and NLRs in pathogen recognition ARTICLE. International immunology, 21(4), 317-337. Koutsky, L.A., Holmes, K.K., Critchlow, C.W., Stevens, C.E., Paavonen, J., Beckmann, A.M., DeRouen, T.A., Galloway, D.A., Vernon, D. and Kiviat, N.B., 1992. A cohort study of the risk of cervical intraepithelial neoplasia grade 2 or 3 in relation to papillomavirus infection. New England journal of medicine, 327(18), 1272-1278. Lam, J.O., Bream, J.H., Sugar, E.A., Coles, C.L., Weber, K.M., Burk, R.D., Wiley, D.J., Cranston, R.D., Reddy, S., Margolick, J.B. and Strickler, H.D., 2016. Association of serum cytokines with oral HPV clearance. Cytokine, 83, 85-91. Lee, S.J., Cho, Y.S., Cho, M.C., Shim, J.H., Lee, K.A., Ko, K.K., Choe, Y.K., Park, S.N., Hoshino, T., Kim, S. and Dinarello, C.A., 2001. Both E6 and E7 oncoproteins of human papillomavirus 16 inhibit IL-18-induced IFN-γ production in human peripheral blood mononuclear and NK cells. The journal of immunology, 167(1), 497-504. Leggatt, G.R. and Frazer, I.H., 2007. HPV vaccines: the beginning of the end for cervical cancer. Current opinion in immunology, 19(2), 232-238. Lin, W.W. and Karin, M., 2007. A cytokine-mediated link between innate immunity, inflammation, and cancer. The Journal of clinical investigation, 117(5), 1175-1183. Mangino, G., Chiantore, M.V., Iuliano, M., Fiorucci, G. and Romeo, G., 2016. Inflammatory microenvironment and human papillomavirus-induced carcinogenesis. Cytokine & growth factor reviews. Mangino, G., Zangrillo, M.S., Chiantore, M.V., Iuliano, M., Accardi, R., Tommasino, M., Fiorucci, G. and Romeo, G., 2015. ID: 109: Pro-inflammatory cytokines and chemokines in HPV-positive cancer cells. Cytokine, 76(1), p.86. Mantovani, F. and Banks, L., 2001. The human papillomavirus E6 protein and its contribution to malignant progression. Oncogene, 20(54), 7874-7887. Mantovani, A., Allavena, P., Sica, A. and Balkwill, F. 2008. “Cancer related inflammation,” Nature, 454(7203), 436-444. McKaig, R.G., Baric, R.S. and Olshan, A.F., 1998. Human papillomavirus and head and neck cancer: epidemiology and molecular biology. Head & neck, 20(3), 250-265. O’Byrne, K.J. and Dalgleish, A.G., 2001. Chronic immune activation and inflammation as the cause of malignancy. British journal of cancer, 85(4), p.473. Ondrey, F.G., Dong, G., Sunwoo, J., Chen, Z., Wolf, J.S., Crowl‐Bancroft, C.V., Mukaida, N. and Van Waes, C., 1999. Constitutive activation of transcription factors NF‐κB, AP‐1, and NF‐IL6 in human head and neck squamous cell carcinoma cell lines that express pro‐inflammatory and pro‐angiogenic cytokines. Molecular carcinogenesis, 26(2), 119-129. Prusty, B.K. and Das, B.C., 2005. Constitutive activation of transcription factor AP‐1 in cervical cancer and suppression of human papillomavirus (HPV) transcription and AP‐1 activity in HeLa cells by curcumin. International journal of cancer, 113(6), 951-960.

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Ray, C.A., Black, R.A., Kronheim, S.R., Greenstreet, T.A., Sleath, P.R., Salvesen, G.S. and Pickup, D.J., 1992. Viral inhibition of inflammation: cowpox virus encodes an inhibitor of the interleukin-1β converting enzyme. Cell, 69(4), 597-604. Richards, K.H., Wasson, C.W., Watherston, O., Doble, R., Blair, G.E., Wittmann, M. and Macdonald, A., 2015. The human papillomavirus (HPV) E7 protein antagonises an Imiquimod-induced inflammatory pathway in primary human keratinocytes. Scientific reports, 5. Rosa, F. and Barnaba, V., 1998. Persisting viruses and chronic inflammation: understanding their relation to autoimmunity. Immunological reviews, 164(1), 17-27. Scott, M., Nakagawa, M. and Moscicki, A.B., 2001. Cell-mediated immune response to human papillomavirus infection. Clinical and diagnostic laboratory immunology, 8(2), 209-220. Shekari, M., Kordi-Tamandani, D.M., MalekZadeh, K., Sobti, R.C., Karimi, S. and Suri, V., 2012. Effect of anti-inflammatory (IL-4, IL-10) cytokine genes in relation to risk of cervical carcinoma. American journal of clinical oncology, 35(6), 514-519. Stanley, M.A., 2012. Epithelial cell responses to infection with human papillomavirus. Clinical microbiology reviews, 25(2), 215-222. Textor, S., Accardi, R., Havlova, T., Hussain, I., Sylla, B.S., Gissmann, L. and Cerwenka, A., 2011. NF‐κ B‐dependent upregulation of ICAM‐1 by HPV16‐E6/E7 facilitates NK cell/target cell interaction. International Journal of Cancer, 128(5), 1104-1113. Tezal, M., 2012. Interaction between chronic inflammation and oral HPV infection in the etiology of head and neck cancers. International journal of otolaryngology, 2012. Tsukui, T., Hildesheim, A., Schiffman, M.H., Lucci, J., Contois, D., Lawler, P., Rush, B.B., Lorincz, A.T., Corrigan, A., Burk, R.D. and Qu, W., 1996. Interleukin 2 production in vitro by peripheral lymphocytes in response to human papillomavirus-derived peptides: correlation with cervical pathology. Cancer research, 56(17), 3967-3974. Xu, Q., Wang, S., Xi, L., Wu, S., Chen, G., Zhao, Y., Wu, Y. and Ma, D., 2006. Effects of human papillomavirus type 16 E7 protein on the growth of cervical carcinoma cells and immunoescape through the TGF-β1 signaling pathway. Gynecologic oncology, 101(1), 132-139. zur Hausen H and Schneider A. 1987. The Papilloma-viruses. Salzman NP, Howley PM (eds). Plenum Publish-ing Corp.: New York, pp. 245-263.

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AIDS: spread the KNOWLEDGE… not the VIRUS Pranveer Singh Indira Gandhi National Tribal University (IGNTU), Amarkantak-484887 (MP), India

Introduction Acquired immune deficiency syndrome or acquired immunodeficiency syndrome (AIDS) is a set of symptoms and infections resulting from the damage to the human immune system caused by the human immunodeficiency virus (HIV) (Fig. 1). This condition progressively reduces the effectiveness of the immune system and leaves individuals susceptible to opportunistic infections and tumors. HIV is transmitted through direct contact of a mucous membrane or the bloodstream with a bodily fluid containing HIV, such as blood, semen, vaginal fluid, pre-seminal fluid, and breast milk. This transmission can involve anal, vaginal or oral sex, blood transfusion, contaminated hypodermic needles, exchange between mother and baby during pregnancy, childbirth, or breastfeeding, or other exposure to one of the above bodily fluids (Blankson, 2010; Sharp and Hann, 2011).

Fig. 1: Structure of HIV

Classification of HIV There are two types of HIV: HIV-1 and HIV-2. Both types are transmitted by sexual contact, through blood, and from mother to child, and they appear to cause clinically indistinguishable AIDS. However, it seems that HIV-2 is less easily transmitted, and the period between initial infection and illness is longer in the case of HIV-2. Worldwide, the predominant virus is HIV-1, and generally when people refer to HIV without specifying the type of virus they will be referring to HIV-1. The relatively uncommon HIV-2 type is concentrated in West Africa and is rarely found elsewhere (Fig. 2). The strains of HIV-1 can be classified into three groups: the “major” group M, the “outlier” group O and the “new” group N. These three groups may represent three separate introductions of simian immunodeficiency virus into humans.

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Group O appears to be restricted to west-central Africa and group N-discovered in 1998 in Cameroon- is extremely rare. More than 90% of HIV-1 infections belong to HIV-1 group M and, unless specified, the rest of this page will relate to HIV-1 group M only (Fig. 2).

Fig. 2: Levels of HIV classification

Within group M there are known to be at least nine genetically distinct subtypes (or clades) of HIV-1. These are subtypes A, B, C, D, F, G, H, J and K (Fig. 2). Occasionally, two viruses of different subtypes can meet in the cell of an infected person and mix together their genetic material to create a new hybrid virus (a process similar to sexual reproduction). Many of these new strains do not survive for long, but those which infect more than one person are known as “circulating recombinant forms” or CRFs. For example, the CRF A/B is a mixture of subtypes A and B. The classification of HIV strains into subtypes and CRFs is a complex issue and the definitions are subject to change as new discoveries are made. Some scientists talk about subtypes A1, A2, A3, F1 and F2 instead of A and F, though others regard the former as sub-subtypes (Fig. 2). The most prominent effect of the HIV virus is its T-helper cell suppression and lysis. The cell is killed off or deranged to the point of being function-less (they do not respond to foreign antigens). The infected B-cells cannot produce enough antibodies either. Thus the immune system collapses leading to the familiar AIDS complications, like infections and neoplasms (Chu and Selwyn, 2011). Although treatments for AIDS and HIV can slow the course of the disease, there is currently no vaccine or cure. Antiretroviral treatment reduces both the mortality and the morbidity of HIV infection, but these drugs are expensive and routine access to antiretroviral medication is not available in all countries. In the absence of antiretroviral therapy, the median time of progression from HIV infection to AIDS is nine to ten years, and the median survival time after developing AIDS is only 9.2 months (Sepkowitz, 2001). WHO disease staging system ƒ Stage I: HIV infection is asymptomatic and not categorized as AIDS

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Stage II: includes minor mucocutaneous manifestations and recurrent upper respiratory tract infections

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Stage III: includes unexplained chronic diarrhea for longer than a month, severe bacterial infections and pulmonary tuberculosis

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Stage IV: includes toxoplasmosis of the brain, candidiasis of the esophagus, trachea, bronchi or lungs and Kaposi’s sarcoma; these diseases are indicators of AIDS (Fig. 3).

Fig. 3: HIV affects almost every organ of the body

Epidemiology, History and Economic impact AIDS is now a pandemic. In 2007, an estimated 33.2 million people lived with the disease worldwide, and it killed an estimated 2.1 million people, including 330,000 children. Over three-quarters of these deaths occurred in sub-Saharan Africa, retarding economic growth and destroying human capital. Most researchers believe that HIV originated in sub-Saharan Africa during the twentieth century. AIDS was first recognized by the U.S. Centers for Disease Control and Prevention in 1981 and its cause, HIV, identified by American and French scientists in the early 1980s (Kallings, 2008; Beyrer et al., 2012).

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Transmission It has been observed that certain subtypes/CRFs are predominantly associated with specific modes of transmission. In particular, subtype B is spread mostly by homosexual contact and intravenous drug use (essentially via blood), while subtype C and CRF A/E tend to fuel heterosexual epidemics (via a mucosal route). Whether there are biological causes for the observed differences in transmission routes remains the subject of debate. Some scientists, such as Dr Max Essex of Harvard, believe such causes do exist. Among their claims are that subtype C and CRF A/E are transmitted much more efficiently during heterosexual sex than subtype B. However, this theory has not been conclusively proven. More recent studies have looked for variation between subtypes in rates of mother-to-child transmission. It has been claimed that such transmission is more common with subtype D than subtype A, and that subtype C is more often transmitted than either D or A (Fig. 4 & 5).

Fig. 4: Modes of HIV transmission

Fig. 5: Myth about HIV transmission

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Associated Infections (Holmes et al., 2003; Sestak 2005) Pulmonary infections: Pneumocystis pneumonia, Tuberculosis (TB) Gastrointestinal infections: Esophagitis, chronic diarrhea, cryptosporidiosis, microsporidiosis Neurological and psychiatric involvement: Toxoplasmosis, Cryptococcal, Progressive multifocal leukoencephalopathymeningitis, AIDS dementia complex (ADC) Tumors and malignancies: Kaposi’s sarcoma (KS), Burkitt’s lymphoma, Cervical cancer, Hodgkin’s disease, anal and rectal carcinomas (Table 1). Misconceptions A number of misconceptions have arisen surrounding HIV/AIDS. Three of the most common are that AIDS can spread through casual contact, that sexual intercourse with a virgin will cure AIDS, and that HIV can infect only homosexual men (anal intercourse between gay men can lead to AIDS infection) and drug users. Cells affected The virus, entering through which ever route, acts primarily on the following cells (Mehandru et al., 2007): •

Lymphoreticular system: o CD4+ T-Helper cells o CD4+ Macrophages o CD4+ Monocytes o B-lymphocytes

•

Certain endothelial cells

•

Central nervous system: o Microglia of the nervous system o Astrocytes o Oligodendrocytes o Neurones-indirectly by the action of cytokines and the gp-120

HIV test HIV tests are usually performed on venous blood using fourth generation screening tests which detect anti-HIV antibody (IgG and IgM) and the HIV p24 antigen. The window period (the time between initial infection and the development of detectable antibodies against the infection) can vary since it can take 3-6 months to seroconvert and to test positive (Chou et al., 2012; Simonetti et al., 2015).

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Prevention Table 1: Estimated per act risk for acquisition of HIV by exposure route Exposure Route

Estimated infections per 10,000 exposures to an infected source 9,000 2,500 67 30 50 6.5 10 5 1 0.5

Blood Transfusion Childbirth Needle-sharing injection drug use Percutaneous needle stick Receptive anal intercourse Insertive anal intercourse Receptive penile-vaginal intercourse Insertive penile-vaginal intercourse Receptive oral intercourse Insertive oral intercourse

Treatment Abacavir- a nucleoside analog reverse transcriptase inhibitor (NARTI or NRTI) There is currently no vaccine or cure for HIV or AIDS. The only known methods of prevention are based on avoiding exposure to the virus or, failing that, an antiretroviral treatment directly after a highly significant exposure, called postexposure prophylaxis (PEP). PEP has a very demanding four week schedule of dosage. It also has very unpleasant side effects including diarrhea, malaise, nausea and fatigue. Antiviral therapy Current treatment for HIV infection consists of highly active antiretroviral therapy, or HAART. Current optimal HAART options consist of combinations (or “cocktails”) consisting of at least three drugs belonging to at least two types, or “classes,” of antiretroviral agents. Typical regimens consist of two nucleoside analogue reverse transcriptase inhibitors (NARTIs or NRTIs) plus either a protease inhibitor or a non-nucleoside reverse transcriptase inhibitor (NNRTI). HAART allows the stabilization of the patient’s symptoms and viremia, but it neither cures the patient of HIV, nor alleviates the symptoms, and high levels of HIV-1, often HAART resistant, return once treatment is stopped. Side effects can also deter people from persisting with HAART, these include lipodystrophy, dyslipidaemia, diarrhoea, insulin resistance, an increase in cardiovascular risks and birth defects. Anti-retroviral drugs are expensive, and the majority of the world’s infected individuals do not have access to medications and treatments for HIV and AIDS (Orrell 2005; Eaton 2007; Beard et al., 2009). Experimental and proposed treatments Most current HIV-1 antiretroviral drug regimens were designed for use against subtype B, and so hypothetically might not be equally effective in Africa or Asia where other strains are more common. At present, there is no compelling evidence that subtypes differ in their sensitivity to antiretroviral drugs. However, some 69 

 

subtypes may occasionally be more likely to develop resistance to certain drugs. In some situations, the types of mutations associated with resistance may vary. This is an important subject for future research. The effectiveness of HIV-1 treatment is monitored using viral load tests. It has been demonstrated that some such tests are sensitive only to subtype B and can produce a significant underestimate of viral load if used to process other strains. The latest tests do claim to produce accurate results for most Group M subtypes, though not necessarily for Group O. It is important that health workers and patients are aware of the subtype/CRF they are testing for and of the limitations of the test they are applying. Not all of the drugs used to treat HIV-1 infection are as effective against HIV2. In particular, HIV-2 has a natural resistance to NNRTI antiretroviral drugs and they are therefore not recommended. As yet there is no FDA-licensed viral load test for HIV-2 and those designed for HIV-1 are not reliable for monitoring the other type. Instead, response to treatment may be monitored by following CD4+ T-cell counts and indicators of immune system deterioration. More research and clinical experience is needed to determine the most effective treatment for HIV-2. Only vaccine can halt the pandemic because a vaccine would possibly cost less, thus being affordable for developing countries, and would not require daily treatments. However, even after several decades of research, HIV-1 remains a difficult target for a vaccine. Researchers have discovered an abzyme that can destroy the protein gp120 CD4 binding site (Walker 2007; Rynell and Trkola 2012). The development of an AIDS vaccine is affected by the range of virus subtypes as well as by the wide variety of human populations who need protection and who differ, for example, in their genetic make-up and their routes of exposure to HIV. In particular, the occurance of superinfection indicates that an immune response triggered by a vaccine to prevent infection by one strain of HIV may not protect against all other strains. The effectiveness of a vaccine is likely to vary in different populations unless some innovative method is developed which guards against many virus strains. Inevitably, different types of candidate vaccines will have to be tested against various viral strains in multiple vaccine trials, conducted in both highincome and developing countries. Alternative medicine Current studies indicate that that alternative medicine therapies, like acupuncture, multivitamin, herns, ayurveda have little effect on the mortality or morbidity of the disease, but may improve the quality of life of individuals afflicted with AIDS (Littlewood and Vanable, 2008; Irlam et al., 2010) Society and culture AIDS stigma exists around the world in a variety of ways, including ostracism, rejection, discrimination and avoidance of HIV infected people; violence against HIV infected individuals or people who are perceived to be infected with HIV;

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Stigma-related violence or the fear of violence prevents many people from seeking HIV testing, returning for their results, or securing treatment, possibly turning what could be a manageable chronic illness into a death sentence and perpetuating the spread of HIV. Often, AIDS stigma is expressed in conjunction with one or more other stigmas, particularly those associated with homosexuality, bisexuality, promiscuity, prostitution, and intravenous drug use. In many developed countries, there is an association between AIDS and homosexuality or bisexuality, and this association is correlated with higher levels of sexual prejudice such as antihomosexual attitudes (Synder et al., 1999; Herek et al., 2002) References Simonetti FR, Dewar R and Maldarelli F (2015). Diagnosis of human immunodeficiency virus infection. In: Bennett JE, Dolin R, Blaser MJ, eds. Mandell, Douglas, and Bennett’s Principles and Practice of Infectious Diseases. Philadelphia, PA: Elsevier Saunders; pp.122. Sepkowitz KA (2001). AIDS-the first 20 years. N Engl J Med 344: 1764-72. Kallings LO (2008). The first postmodern pandemic: 25 years of HIV/AIDS. J Int Med 263: 21843. Sharp PM and Hahn BH (2011). Origins of HIV and the AIDS Pandemic. Cold Spr Harb Persp Med 1: a006841. Blankson JN (2010). Control of HIV-1 replication in elite suppressors. Discov Med 9: 261-6. Walker BD (2007). Elite control of HIV Infection: implications for vaccines and treatment. Topics in HIV medicine: A publication of the International AIDS Society, USA.15: 134-6. Holmes CB, Losina E, Walensky RP, Yazdanpanah Y and Freedberg KA (2003). Review of human immunodeficiency virus type 1-related opportunistic infections in sub-Saharan Africa. Clin Infect Dis 36: 656-662. Chu C and Selwyn PA (2011). Complications of HIV infection: a systems-based approach. Am Fam Phy 83: 395-406. Sestak K (2005). Chronic diarrhea and AIDS: insights into studies with non-human primates. Curr HIV Res 3: 199-205. Beyrer C, Baral SD, van Griensven F, Goodreau SM, Chariyalertsak S, Wirtz AL and Brookmeyer R (2012). Global epidemiology of HIV infection in men who have sex with men. Lancet 380: 367-77. Mehandru S, Poles MA, Tenner-Racz K, Horowitz A, Hurley A, Hogan C, Boden D, Racz P and Markowitz M (2007). Primary HIV-1 infection is associated with preferential depletion of CD4+ T cells from effector sites in the gastrointestinal tract. J Exp Med 6: 761-70. Eaton LA and Kalichman S (2007). Risk compensation in HIV prevention: implications for vaccines, microbicides, and other biomedical HIV prevention technologies. Curr HIV/AIDS Rep 4: 165-72. Chou R, Selph S, Dana T, et al. (2012). Screening for HIV: systematic review to update the 2005 U.S. Preventive Services Task Force recommendation. Annal Int Med 157: 706-18. Beard J, Feeley F and Rosen S (2009). Economic and quality of life outcomes of antiretroviral therapy for HIV/AIDS in developing countries: a systematic literature review. AIDS Care 21: 1343-56. Orrell C (2005). Antiretroviral adherence in a resource-poor setting. Curr HIV/AIDS Rep 2: 171-6. Nachega JB, Marconi VC, van Zyl GU, Gardner EM, Preiser W, Hong SY, Mills EJ and Gross R (2011). HIV treatment adherence, drug resistance, virologic failure: evolving concepts. Infect Disord Drug Targ 11: 167-74.

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Snyder M, Omoto AM and Crain AL (1999). Punished for their good deeds: stigmatization for AIDS volunteers. Am Behav Sc 42: 1175-1192. Herek GM, Capitanio JP and Widaman KF (2002). HIV-related stigma and knowledge in the United States: prevalence and trends, 1991-1999. Am J Pub Health 92: 371-7. Irlam JH, Visser MM, Rollins NN and Siegfried N (2010). Irlam JH ed. Micronutrient supplementation in children and adults with HIV infection. Cochrane database of systematic reviews (Online) (12): CD003650. Littlewood RA and Vanable PA (2008). Complementary and alternative medicine use among HIVpositive people: research synthesis and implications for HIV care. AIDS Care 20: 1002-18. Reynell L and Trkola A (2012). HIV vaccines: an attainable goal? Swiss Med Weekly 142: w13535.

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OIE recommended nucleic acid based techniques for poultry diseases diagnosis Manas Ranjan Prabhraj, Kuldeep Dhama and Deepak Kumar Division of Veterinary Biotechnology, ICAR-Indian Veterinary Research Institute, Izatnagar-243 122 (UP)

Introduction Poultry is one of the fastest growing segments amongst all the Indian agricultural sectors. While the production of agricultural crops has been rising at a rate of 1.5 to 2 percent per annum, the eggs and broilers has been rising at a rate of 8 to 10 percent per annum. Currently, India is the world’s fifth largest egg and the eighteenth largest broilers producer. The Potential in the sector is due to several factors including-growth in per capita income, a growing urban population, increase demand of poultry and falling real poultry prices. Poultry meat is the fastest growing component of global meat demand. In India, poultry sector growth is being driven by rising incomes and a rapidly expanding middle class, together with the emergence of vertically integrated poultry producers that have reduced consumer prices by lowering production and marketing costs. The country has exported 6,59,304.15 MT of Poultry products to the world for the worth of Rs. 768.72 crores during the year 2015-16. However, there are some factors which constitute major constraints like low genetic potential, poor nutrition, low productivity of some indigenous stock and high incidence of infectious diseases to the development and improvement of the poultry industry in India. Most importantly large poultry population and poultry industry is jeopardizing by various infectious diseases. To minimize the losses incurred to poultry sector by hazardous infectious disease, quick and reliable diagnostic procedures are the key. Many traditional laboratory methods have already been developed for the diagnosis like serum neutralization immunodiffusion, immunofluorescence, agar gel precipitation test (AGPT), indirect fluorescent antibody (IFA) assay, virus neutralization (VN) assay, Agar gel immuno-diffusion test (AGID) Serum neutralization test (SN), and enzyme-linked immunosorbent assay. Nucleic acid-based testing is becoming a crucial diagnostic tool for a wide variety of infectious diseases in poultry. Following diagnosis, molecular testing can help guide appropriate prevention and control strategy. Successful application biologic response modifiers and assessing disease prognosis and therapy response and detecting minimal residual disease are offered by molecular diagnostics. As many developments in molecular techniques towards diagnosis have made, molecular diagnostics is becoming an integral part of poultry disease diagnosis. In the current review we summarize the OIE recommended molecular diagnosis procedures in OIE listed poultry diseases. PCR and RT PCR: The purpose of a PCR (Polymerase Chain Reaction) is to amplify a gene to produce huge number of copies. This is necessary to have enough starting template for diagnostic and sequencing. With three major steps and 30 or 40

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cycles PCR is done on an automated cycler, which can heat and cool the tubes with the reaction mixture in a very short time. PCR has been found to be more sensitive than virus isolation for clinical samples of Chlamydophila psittaci. PCR is a test of choice for the Avian infectious laryngotracheitis Virus (ILTV) (Humberd et al., 2002). Fowl Pox virus (Fallavena et al., 2002; Lee et al., 1997) and Marek’s Disease (MD). For detection of C. psittaci PCR is done to target the ompA gene or the 16S23S rRNA gene (Sachse et al., 2009). RT PCR i.e. reverse transcriptase PCR can also be used to amplify an RNA target sequence. By using a reverse transcriptase enzyme from a retrovirus, the RNA sequence is first converted to a double-stranded nucleic acid sequence (cDNA). The cDNA sequence can then be amplified by using the same PCR cycles already described. RT-PCR is used for detection of RNA viruses viz. Avian Infectious Bronchitis Virus (Gelb et al., 2005). Due to fragile nature of RNA fresh samples are generally required for RT-PCR technique which helps in rapid detection and subtype identification of H5 and H7 Avian Flu with the correctly defined primers. This cDNA product can be used for nucleotide sequencing for type identification of Avian Flu virus (Suarez, 2007). This technique was used with success during the 2003 HPAI outbreaks in The Netherlands. For NDV usually RT-PCR is used to amplify a specific portion of the genome (like part of the F gene that contains the F0 cleavage) site so that the product can be used for assessing virulence also (Creelan et al., 2002). RT-PCR genotyping methods have largely replaced HI and VN serotyping for determining the identity of afield strain of NDV. REAL TIME PCR: The recent development of Real-Time PCR (rRT-PCR) added great advantages to traditional PCR by allowing real-time quantitation of PCR product by measuring the amount of fluorescence emitted from a dye intercalated in the double-helix DNA product after each amplification cycles and the amount of fluorescence is directly proportional to the number of copies after amplification of target, but after reaching a certain critical copy number the amount of fluorescence increases by an exponential amount. As depicted in the fluorescence vs cycle number plot in the cycle in which the critical copy number is reached is directly dependent on how many target DNA copies were present in the original sample before any amplification. There is less chance of contamination as the entire process of amplification and quantitation of the original target DNA for each sample is done in a single sealed tube. One of the strategies used to avoid post-amplification processing is to employ real-time RT-PCR (rRT-PCR) techniques. The advantages of such assays are that rRT-PCR assays based on the fluorogenic hydrolysis probes or ‘TaqMan’ method or fluorescent dyes eliminate the post-amplification processing step and that results can be obtained in less than 3 hours. Forat least partialdiagnosis it has become the most suitable method in many laboratories from clinical specimens directly. With sensitivity and specificity comparable to virus isolation this offers rapid result. Spackman et al. (2002) detected Avian Influenza viruses and determined subtype H5 or H7by using a single-step rRT-PCR primer/fluorogenic hydrolysis probe system.

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The test offered a cheaper and much more rapid alternative, with diagnosis on clinical samples in less than 3 hours as compared to virus isolation. rRT-PCR is recommended by OIE for Infectious Laryngio Trachitis Virus (ILTV) (Creelan et al., 2006), to quantify MDV genome copies (Abdul-Careem et al., 2006), Chlamydophila psittaci (Pantchev et al., 2009). Differentiation of oncogenic and non-oncogenic strains of serotype 1 MDV, and of MDV vaccine strains of serotypes 2 and 3 are performed in rRT-PCR (Handberg et al., 2001). rRT-PCR may also be used to quantify virus load in tissues (Baigent et al., 2005) or differentially diagnose MDV and HVT in the blood or feather tips (Baigent et al., 2005). The widest application of an rRT-PCR assay for APMV-1 detection was in the United States of America (USA) during the Newcastle Disease outbreaks of 2002-2003, when the assay described by Wise et al. (2004) was employed.

LAMP PCR: LAMP is isothermal nucleic acid amplification. Isothermal amplification in general obviates the need for thermal cyclers. With color change by addition of SYBR green or by seeing turbidity via photometry with an increasing quantity of Magnesium pyrophosphate in solution, detection of amplification product can be determined. Also in-tube detection of DNA amplification is possible 75 

 

with fluorescence complex formation of manganese by pyrophosphate by using manganese loaded calcein during in vitro DNA synthesis. Primers for LAMP are designed from the target gene of the pathogen. All primers for LAMP are designed as described by Notomi et al. (2000). Of the two inner primers of LAMP, each primer has two binding regions connected by a TTTT spacer. Primer Explorer programme on website http://primerexplorer.jp/ will be used to design the specific primers. This software generates the four primer sets containing FIP (Forward Inner Primer), F3, BIP (Backward Inner Primer) and B3 based on the target sequence information. FIP (BIP) consists of the sequence of the F1c (B1c) and F2 (B2) regions and F1, F2, F3 of 20bp long sequences are selected from the target gene, B1, B2, B3 of 20bp long sequences are selected from the complementary strand. Primers from complementary regions are F1c and F1, B1 and B1c. Recently a Real Time Lamp strategy is devised for real time detection and quantification of ARV (Kumar et al., 2016, Article in press). MICRO ARRAY: Using DNA microarrays for detection of pathogen holds great promise for the future of molecular diagnostics. This technology allows, in one assay, for simultaneous assessment and detection of pathogen from an unknown sample with several infections. Oligonucleotide/DNA chip which is asilicon chip with sequences of 12,000 or more genesis used as the immobilized probes and the complementary specific mRNA will hybridize to it.DNA microarray technology as diagnostic method was recently used for chlamydial infection (Sachse et al., 2005). Due to its high sensitivity and specificity as comparable to real-time PCR it has been proved suitable for routine diagnosis for example in this sample DNA is hybridised to 36 Chlamydia specific oligonucleotide probes. This methodological approach enables detection of mixed chlamydial infections and identification of unexpected and unknown chlamydial species directly from clinical samples (Borel et al., 2008). Gene Targeted Sequencing: Nucleotide sequencing of a diagnostically relevant fragment of the gene the differentiation of Infectious Bronchitis Virus (IBV) strains and has become the genotyping method of choice in many laboratories. RT-PCR product cycle sequencing of the hypervariable amino terminus region of S1 may be used diagnostically to identify previously recognized field isolates and variants of IBV (Kingham et al., 2000). Gene-targeted sequencing (GTS), is a technique which uses the sequence of a particular gene for identification of a pathogen. PCR primers are used for the mgc2, gapA, pvpA, and MGA_0309 genes of Mycoplasma gallisepticum can be used to provide an accurate and reproducible method of typing of strains, which will help towards rapid global comparisons between laboratories (Ferguson et al., 2005). Preliminary strain identification by sequencing of the PCR product which is obtained by taking PCR primers for the mgc2 gene of Mycoplasma gallisepticum or the vlhA gene of Mycoplasma synovae allows for preliminary strain identification without the need for prior isolation of the organism (Hong et al., 2004; 2005). PCR RFLP: Restriction Fragment Length Polymorphism (RFLP) is a difference in homologous DNA sequences after digestion of the DNA samples with specific

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restriction endonucleases that can be detected by the presence of fragments of different lengths. RFLP, as a molecular marker, is specific to a single clone/ restriction enzyme combination and mostly they are co-dominant (both alleles in heterozygous sample are there) and are highly locus-specific. A labeled DNA sequence i.e. RFLP probe hybridizes with one or more fragments of the digested DNA sample after they were separated by gel electrophoresis, thus revealing a unique blotting pattern characteristic to a specific genotype at a specific locus. Short, DNA or cDNA clones are typically used as RFLP probes. The primary advantages of genotyping methods are a rapid turnaround time, and the ability to detect a variety of genotypes, depending on the tests used. RFLP RT-PCR differentiates various serotypes based on unique electrophoresis banding patterns of restriction enzymedigested fragments of target gene following amplification of the gene by RTPCR.OIE recommends use of RFLP in cases of IBV (Jackwood et al., 1997). A range of restriction endonucleases (RE) have been described in for RFLP analysis of ILTV PCR products and several genes like ICP4, TK (thymidine kinase), UL 15, UL47 glycoprotein G and ORF-BTK have been targeted for digestion. The combination of PCR and RFLP helps to differentiate field strains of ILTV from vaccine strains (Ojkic et al., 2006). RFLP probes from IS901, IS1245 and IS1311 are used to investigate the molecular epidemiology of M. avium and M. intracellulare infection, in humans (O’grady et al., 2000). Restriction fragment length polymorphism (RFLP) analysis can be used to differentiate field isolates from vaccine strains of fowl pox virus (Ghildyal et al., 1989; Schnitzlein et al., 1988). Standard molecular ‘fingerprinting’ techniques like plasmid profile analysis, pulsed field gel electrophoresis, PCR-restriction fragment length polymorphism (RFLP) or ribotyping are used for investigating outbreaks of S. Pullorum or S. Gallinarum. Most efforts at molecular identification have given for the characterisation of the larger segment of IBDV (segment A) especially of the vVP2 encoding region (Zierenberg et al., 2001). Nucleic Acid Sequence-based Amplification (NASBA ASSAY): Avian myeloblastosis virus (AMV), reverse transcriptase (RT), T7 RNA polymerase and RNase H with two oligonucleotide primers are required for NASBA reaction where the amplification is more than 1012 fold within 90 to 120 minutes. As it is an isothermal process the amplification of ssRNA is possible only when the denaturation of dsDNA does not occur. Unlike RT-PCR the NASBA reaction does not get false positives caused by genomic dsDNA. A fluorescence signal during the amplification is occurred in the NASBA due to binding of beacon with amplified RNA. For example, (NASBA) with electrochemiluminescent detection (NASBA/ ECL) is a continuous isothermal reaction in which specialized thermo cycling equipment is not required. The detection of AIV subtypes H7 and H5 in clinical samples within 6 hours can be done by NASBA assays (Ko et al., 2004). RAPD: DNA fragments from PCR amplification of random segments of genomic DNA with single primer of arbitrary nucleotide sequence are called as Random Amplified Polymorphic DNA (RAPD) markers. Unlike traditional PCR analysis, there is no requirement of prior knowledge of DNA sequence of the target organism 77 

 

in RAPD: depending on positions on target DNA that are complementary to the primers’ sequences, identical 10-mer primers will or will not amplify a segment of DNA. For example, if primers annealed too far apart or 3’ ends of the primers are not facing each other there is no production of fragment. Therefore, there should not be any mutation in template DNA which will give arbitrary pattern of amplified DNA on the gel as there is no annealing of primer to complementary strand. Arbitrary primed PCR or random amplified polymorphic DNA (RAPD) is required for accurate method for doing DNA fingerprinting. This technique uses short, arbitrary PCR primers, which generate reproducible patterns in agarose gels (Fan et al., 1995). For rapid identification of strains of Mycoplasma gallisepticum for epidemiological studies this method is rapid and simple. Random amplified polymorphic DNA (RAPD) hasproved particular useful in study of Salmonella gallinarum (Habtamu-Taddele et al., 2011). Nucleic Acid Hybridization Probes/Fish: In FISH a fluorescence-labeled oligonucleotide probes specifically attach to their complementary DNA sequence target on the genome and label that region with fluorescence color (e.g., Texas red). The labeled region can then be easily visualized under a fluorescence microscope. Study of chromosomal deletions and translocations and gene amplifications can be done by FISH which is a good as compared to conventional epigenetic method. As per OIE guidelines commercial nucleic acidhybridisation probes have become a ‘gold standard’ for distinction between M. avium and M. intracellulare cultures. M. genavense can also be distinguished with these tests. A further probe that covers the whole MACwas also developed, as genuine MAC strains have been described that fail to react with specific M. avium and M. intracellular probes (Sony et al., 1996). These tests use a chemiluminescent-labelled, single-stranded DNA probe that iscomplementary to the ribosomal RNA of the target organism. The labelled DNARNA hybrids are measured in aluminometer. Cloned genomic fragments of fowl pox virus can be used effectively as nucleic acid probes for diagnosis off owlpox. Viral DNA isolated from lesions can be detected by hybridisation either with radioactively or non-radioactively labelled genomic probes. This method is especially useful for differentiation of fowlpox from infectious laryngotracheitis when tracheal lesions are present (Fatunmbi et al., 2005). High Throughput Sequencing: After capillary sequencing methods that relied upon Sanger sequencing, Next-generation sequencing (NGS) comes in the last decade for DNA sequencing. Unlike the Sanger method i.e. chain-termination sequencing, NGS methods enable the sequencing of thousands to millions of molecules at once as highly parallelized manner. Popular NGS methods include pyrosequencing which makes use of luciferase to read out signals as individual nucleotides are added to DNA templates developed by Roche Life Sciences, Illumina sequencing which uses reversible dye-terminator techniques that adds a single nucleotide to the DNA template in each cycle and SOLID sequencing which sequences by preferential ligation of fixed-length oligonucleotides developed by Life Technologies. As per OIE guidelines High throughput sequencing has also been applied to S. gallinarum,

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but is not yet economically viable for outbreak investigation (Richardson et al., 2011). As many molecular techniques have made their expected transitions into the clinical arena, molecular diagnostics is becoming an integral part of poultry practice. Molecular diagnostics together with genomic-based approach, when combined with have potential to deliver valuable information on disease outbreak well in time. Therefore, these approaches can be employed to provide most desired information on host pathogen interaction and to develop suitable strategy to combat with infectious diseases of poultry. References Abdul-Careem M.F., Hunter B.D., Nagy E., Read L.R., Sanei B., Spencer J.L. and Sharif S. (2006). Development of a real-time PCR assay using SYBR Green chemistry for monitoring Marek’s disease virus genome load in feather tips. J. Virol. Methods, 133(1): 34-40. Baigent S.J., Petherbridge L.J., Howes K., Smith L.P., Currie R.J.W. and Nair V. (2005). Absolute quantitation of Marek’s disease virus genome copy number in chicken feather and lymphocyte samples using real-time PCR. J.Virol. Methods, 123, 53-64. Borel N., Kempf E., Hotzel H., Schubert E., Torgerson P., Slickers P., Ehricht R., Tasara T., Pospischil A. and Sachse K. (2008). Direct identification of chlamydiae from clinical samples using a DNA microarray assay-a validation study. Mol. Cell. Probes, 22: 55-64. Creelan J.L., Calvert V.M., Graham D.A. and Mcculloch J. (2006). Rapid detection and characterisation from field cases of infectious laryngotracheitis by real-time polymerase chain reaction and restriction fragment length. Avian Pathol., 35, 173-179. Creelan J.L., Graham D.A. and Mccullough S.J. (2002). Detection and differentiation of pathogenicity of avian paramyxovirus serotype 1 from field cases using one-step reverse transcriptase-polymerase chain reaction. Avian Pathol.,31 (5), 493-499. Eterradossi N., Arnauld C., Toquin D. and Rivallan G. (1998). Critical amino acid changes in VP2 variable domain are associated with typical and atypical antigenicity in very virulent infectious bursal disease viruses. Arch. Virol., 143, 1627-1636. Fallavena L.C., Canal C.W., Salle C.T., Moraes H.L., Rocha S.L. and Pereira Da Silva A.B. (2002). Presence of avipoxvirus DNA in avian dermal squamous cell carcinoma.Avian Pathol., 31: 241-246. Fan H.H., Kleven S.H. and Jackwood M.W. (1995). Application of polymerase chain reaction with arbitrary primers to strain identification of Mycoplasma gallisepticum. Avian Dis., 39: 729735. Fatunmbi O.O., Reed W.M., Schwartz D.I. and Tripathy D.N. (1995). Dual infection of chickens with pox and infectious laryngotracheitis (ILT) confirmed with specific pox and ILT DNA dot-blot hybridization assays. Avian Dis., 39, 925-930. Ferguson N.M., Hepp D., Sun S., Ikuta N., Levisohn S., Kleven S.H. and García M. (2005). Use of molecular diversity of Mycoplasma gallisepticumby gene-targeted sequencing (GTS) and random amplified polymorphic DNA (RAPD) analysis for epidemiological studies Microbiol., 151: 1883-1893. Gelb J., JR., Wolff J.B. and Moran C.A. (1991). Variant serotypes of infectious bronchitis virus isolated from commercial layer and broiler chickens. Avian Dis., 35: 82-87. Ghildyal N., Schnitzlein W.M. and Tripathy D.N. (1989). Genetic and antigenic differences between fowlpox and quailpox viruses.Arch. Virol., 106: 85-92.

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Habtamu-Taddele M., Rathore R., Dhama K. and Agarwal R.K. (2011). Epidemiological characterization of Salmonella gallinarum Isolates of poultry origin in India, employing two PCR based typing methods of RAPD-PCR and PCR-RFLP. Asian J. Anim. Vet. Adv., 6: 1037-1051. Handberg K.J., Nielson O.L. and Jorgensen P.H. (2001). Use of serotype 1 & serotype 3 specific polymerase chain reaction for the detection of Marek’s disease virus in chickens. Avian Pathol.,30, 243-249. Hong Y., García M., Leiting L., Bencina D., Dufour-Zavala L., Zavala G. and Kleven S.H. (2004). Specific detection and typing of Mycoplasma synoviaestrains in poultry with PCR and DNA sequence analysis targeting the hemagglutinin encoding gene VLHA. Avian Dis., 48, 606616. Hong Y., Garcia M., Levisohn S., Savelkoul P., Leiting V., Lysnyansky I., Ley D.H. and Kleven S.H. (2005). Differentiation of Mycoplasma gallisepticumstrains using amplified fragment length polymorphism and other DNA-based typing methods. Avian Dis., 49, 43-49. Humberd J., Garcia M., Riblet S.M., Resureccion R.S. and Brown T.P. (2002). Detection of infectious laryngotracheitis virus in formalin-fixed, paraffin-embedded tissues by nested polymerase chain reaction. Avian Dis., 46, 64-74. Imai M., Ninomiya A., Minekawa H., Notomi T., Ishizaki T., Tashiro M. and Odagiri T. (2006). Development of H5 RTLAMP (loop-mediated isothermal amplification) system for rapid diagnosis of H5 avian influenza virus infection. Vaccine, 24: 6679-6682. Jackwood M.W., Yousef N.M. and Hilt D.A. (1997). Further development and use of a molecular serotype identification test for infectious bronchitis virus. Avian Dis., 41, 105-110. Kingham B.F., Keeler C.L. JR, NIX W.A., Ladman B.S. and Gelb J. JR (2000). Identification of avian infectious bronchitis virus by direct automated cycle sequencing of the S-1 gene. Avian Dis., 44, 325-335. Kingham B.F., Keeler C.L. JR, NIX W.A., Ladman B.S. and Gelb J. JR (2000). Identification of avian infectious bronchitis virus by direct automated cycle sequencing of the S-1 gene.Avian Dis., 44, 325-335. Ko L.S., Lau L.T., Banks J., Aherne R., Brown I.H., Collins R.A., Chan K.Y., Xing J. and Yu A.C.H. (2004). Nucleic acid sequence-based amplification methods to detect avian influenza virus.Biochem. Biophys. Res. Commun., 313: 336-342. Lee L.H. and Lee K.H. (1997). Application of the polymerase chain reaction for the diagnosis of fowlpox virus infection. J. Virol. Methods, 63: 113-119. O’grady D., Flynn O., Costello E., Quigley F., Gogarty A., Mcguirk J., O’rourke J. and Gibbons N. (2000). Restriction fragment length polymorphism analysis of Mycobacterium aviumisolates from animal and human sources. Int. J. Tuberc. Lung Dis., 4, 278-281. Ojkic D., Swinton J., Vallieres M., Martin E., Shapiro J., Sanei B. and Binnington B. (2006). Charcterisation of field isolates of infectious laryngotracheitis virus from Ontario. Avian Pathol., 35: 286-292. Pantchev A., Sting R., Bauerfeind R., Tyczka J. and Sachse K. (2009). New real-time PCR tests for species specific detection of Chlamydophila psittaci and Chlamydophila abortus from tissue samples. Vet. J., 181, 145-150. Postel A., Letzel T., Frischmann S., Grund C., Beer M. and Harder T. (2010). Evaluation of two commercial loopmediated isothermal amplification assays for detection of avian influenza H5 and H7 hemagglutinin genes. J. Vet. Diagn. Invest., 22: 61-66. Richardson E.J., Limaye B., Inamdar H., Datta A., Manjari K.S., Pullinger G.D., Thomson N.R., Joshi R.R., Watson M. and Stevens M.P. (2011). Genome sequences of Salmonella enterica sero var typhimurium, Choleraesuis, Dublin, and Gallinarum Strains of well-defined virulence in food-producing animals. J. Bacteriol., 193 (12), 3162-3163.

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Sachse K., Hotzel H., Slickers P., Ellinger T. and Ehricht R. (2005). DNA microarray-based detection and identification of Chlamydia and Chlamydophila. Mol. Cell Probes, 19: 41-50. Sachse K., Vretou E., Livingstone M., Borel N., Pospischil A. and Longbottom D. (2009b). Recent developments in the laboratory diagnosis of chlamydial infections (review). Vet. Microbiol., 135: 2-21. Schnitzlein W.M., Ghildyal N. and Tripathy D.N. (1988).Genomic and antigenic characterisation of avipoxviruses. Virus Res., 10: 65-76. Singh P., Kim T.J. and Tripathy D.N. (2003).Identification and characterisation of fowlpox virus strains using monoclonal antibodies.J. Vet. Diagn.Invest., 15, 50-54. Soini H., Eerola E. and Viljanen M.K. (1996). Genetic diversity among Mycobacterium aviumcomplex Accu-Probe-positive isolates. J. Clin. Microbiol., 34: 55-57. Spackman E., Senne D.A., Myers T.J., Bulaga L.L., Garber L.P., Perdue M.L., Lohman K., Daum L.T. and Suarez D.L. (2002). Development of a real-time reverse transcriptase PCR assay for type A influenza virus and the avian H5 and H7 hemagglutinin subtypes. J. Clin. Microbiol., 40: 3256-3260. Suarez D.L., Das A. and Ellis E. (2007). Review of rapid molecular diagnostic tools for avian influenza virus. Avian Dis., 51, 201-208. Wise M.G., Suarez D.L., Seal B.S., Pedersen J.C., Senne D.A., King D.J., Kapczynski D. and Spackman E. (2004). Development of a real-time reverse-transcription PCR for detection of Newcastle disease virus RNA in clinical samples. J. Clin. Microbiol., 42: 329-338. Zierenberg K., Raue R. and Muller H. (2001). Rapid identification of very virulent strains of infectious bursal disease virus by reverse transcription-polymerase chain reaction combined with restriction enzyme analysis. Avian Pathol., 30: 55-62.

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Brucellosis- a veterinary perspective Himani Dhanze, R.S. Rathore, M. Suman Kumar, K. Dhama and Ashok Kumar ICAR-Indian Veterinary Research Institute, Izatnagar-243 122 (UP), India

Introduction Brucellosis, an important direct zoonosis has been a potent threat to human and animal health with worldwide prevalence. It is among the most ancient diseases described and has been studied since the time of Hippocrates. Brucellosis has an economically devastating effect on the productivity of livestock due to reproductive failure and on mankind due to loss of man-days and labour (Benkirane, 2006). The disease acts as a major deterrent to trade and export of livestock and their products (Radostits et al., 2000). In India, the presence of brucellosis was first established during the early previous century and has since been reported from almost all the states (Renukaradhya et al., 2002). Brucellosis is readily transmissible to humans, and causes acute febrile illness and undulant fever which may progress to a more chronic form and can also produce serious complications affecting the musculo-skeletal, cardiovascular, and central nervous systems. Infection is often due to occupational exposure and is acquired by the oral, respiratory, or conjunctival routes. Ingestion of dairy products constitutes the main risk to the general public where the disease is endemic. There is an occupational risk to veterinarians and farmers who handle infected animals and aborted fetuses or placentas. Brucellosis, classified as a risk group III pathogen as per WHO, is one of the most easily acquired laboratory infections, and strict safety precautions need to be observed when handling cultures and heavily infected samples, such as products of abortion (OIE, 2009). Animal brucellosis is endemic worldwide and bovine brucellosis, caused by B. abortus, remains the most widespread form in animals. Brucellosis causes considerable economic losses through reduced productivity, abortions and birth of weak offspring in livestock. The productivity losses due to B. melitensis infection are not very well documented in Asia. B. melitensis infections usually lead to outbreaks in comparison to a regular endemic pattern, as is frequently found with B. abortus infections (Benkirane, 2006). Brucellosis in pigs has productivity and economic impacts but there is scant data on their epidemiology in low income countries. The predominant route of exposure among animals is through ingestion or inhalation of organisms that are present in fetal fluids or other birth products. Introduction of infected animals to herds is among the main source for disease spread owing to pasture or water contamination from aborted foetus. Reports reveal that transient disease can also develop following administration of a live Brucella vaccine, particularly the B. abortus vaccine strain (Paulsen et al., 2002). Among dogs and sheep, transmission of rough strains of Brucella may be more common via the venereal route, although supporting data are limited. Among dogs, the urine of

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males and vaginal secretions of females are the main sources of infection via the venereal, oral, nasal, or conjunctival routes (Wanke, 2004). Brucellosis in animals and modes of transmission Brucellosis leads to a sub-acute or chronic condition and affects multiple species of animals. In cattle, sheep, goats, other ruminants and pigs the initial phase following infection is often inapparent. In sexually mature animals the infection localizes in the reproductive system and typically produces placentitis followed by abortion in the pregnant female, mostly during the third trimester of pregnancy. In males epididymitis and orchitis are observed. Bovine brucellosis is more commonly caused by B. abortus, less frequently by B. melitensis, and occasionally by B. suis (Robinson, 2003). Infection spreads rapidly and causes abortion ‘storms’ in herds of unvaccinated cattle. B. melitensis is the main cause of brucellosis in sheep and goats and B. suis in swine. Transmission occurs by direct contact and environmental contamination following abortion. Sexual transmission and/or artificial insemination are also common routes of acquiring infection; however, venereal transmission from infected bulls to susceptible cows appears to be rare. Transmission during artificial insemination occurs more commonly when Brucella-contaminated semen is deposited in the uterus but the chances of infection are reduced when semen is deposited in the midcervix. Infections in sheep and goats are highly contagious due to the pathogenicity of B. melitensis and close contact due to the high density of the flocks. Interference with fertility is usually temporary and most infected animals will abort only once and some are unaffected. The udder is often permanently infected, especially in case of cattle and goats. Shedding of organisms in milk is frequent. Localized infections in sheep result in orchitis or epididymitis in the case of B. melitensis and B. ovis. In horses, local abscess formation in bursae may be the only clinical sign and infection in this species is often asymptomatic. The severity of the disease depends upon many factors such as previous vaccination, age, sex and management such as herd or flock size and density. Animal-to-animal transmission occurs as a result of the large number of organisms shed in the environment. Most infections result from ingestion of bacteria either from diseased animals or contaminated feedstuffs. However, infection may also be acquired by respiratory exposure and by contamination of abraded skin and mucosal surfaces. Natural breeding transmits infection in swine and dogs and, to a lesser extent, sheep and goats. B. abortus and B. suis have been isolated from a variety of wildlife species, whereas B. melitensis is rarely reported in wildlife. The interactions between wildlife and livestock, may be the most important drivers for transmission. Till date no vaccine against brucellosis has demonstrated satisfactory safety and efficacy in wildlife, which suggests that controlling brucellosis in wildlife should be based on good management practices. Transmission of Brucella spp. from wildlife to humans is linked to butchering of meat and dressing of infected wild pig or buffalos carcasses (Godfroid et al., 2013).

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Incidence of Brucellosis in livestock in India The exact problem of brucellosis in animals has not been estimated systematically and only scattered reports are available on prevalence of the disease from various parts of the country. The available epidemiological evidences suggest that brucellosis is prevalent in all domestic animals besides wild animals in almost all the states of India with wide variation in prevalence (Boral et al., 2009). A national survey of bovine brucellosis from 1994-2001 recorded a national average of 5% sero-prevalence of brucellosis in cattle (Renukaradhya et al., 2002). This survey indicated a sero-prevalence of 23% in Punjab, 16% in Gujarat and 6.3%, 2.4% and 1.7% in states of Goa, Maharashtra and Andhra Pradesh, respectively. In general, higher prevalence has been reported among cattle than buffaloes, with increasing trend (Maanasi, 1999). The incidence of brucellosis in cattle and buffaloes was reported to be 5% and 3%, respectively, while it was found to be 7.9% in sheep and 2.2% in goats (Renukaradhya et al., 2002). With a sizable population of sheep and goat in the country, prevalence of brucellosis in small ruminants is significant. Shome et al. (2008) reported an overall prevalence of brucellosis in both sheep and goats as 9.95%. The same study reported that the prevalence of the disease was found highest in the state of Gujarat (26.08%) followed by Karnataka (14.93%) and lowest in Rajasthan (5.53%). These results show that brucellosis is endemic at lower level in sheep and goats but lesser in prevalence than that in cattle. Brucellosis has also been detected in mithun (Rajkhowa et al., 2005) and yaks (Bandyopadhyay et al., 2009). Different biotypes have been isolated from various livestock species in India. B. abortus biotypes I and III have been isolated from cattle (Chatterjee et al., 1995), apart from biotype IV (Batra et al., 1989). While biotype I of B. abortus predominates in the cattle in organized dairy farm, B. abortus biotype III has been the predominant biotype in cattle under traditional husbandry systems consisting of both small holdings dispersed in villages and nomadic or semi-nomadic herds (Anon., 1986). Occasionally, isolation of B. melitensis biotype I has also been reported in cattle (Hemshettar et al., 1987). Besides B. abortus biotype I (Panjarathan and Gulrajani, 1974), all biotypes of B. melitensis i.e., biotypes I, II and III have been isolated from sheep and goats (Sharma et al., 1995) with predominance of biotype I (Sen and Sharma 1972). Pillai et al. (1991) isolated B. canis from dog in Chennai. B. suis biotype II has been isolated from a cow in Tamil Nadu (Kumar and Rao 1980). Both B. abortus and B. melitensis have been isolated from horses (Polding, 1947). Brucellosis in humans Brucellosis is a severe and debilitating zoonotic disease in man and manifests as an acute or sub-acute febrile illness usually marked by an intermittent or remittent fever accompanied by malaise, anorexia and prostration, which may persist for weeks or months in the absence of treatment. An estimated 500,000 human infections occur annually worldwide. In order to eradicate or at least control disease, good surveillance, reliable tests and efficacious vaccines are of paramount

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importance. Current tests, although useful, are constrained through problems of both sensitivity and specificity. The possible means of acquisition of brucellosis include: person-to-person transmission, infection from a contaminated environment, occupational exposure usually resulting from direct contact with infected animals, and foodborne transmission. Person-to-person route seems to be extremely rare with circumstantial evidence suggests close personal or sexual contact as the route of transmission. Inhalation brucellosis may then result from exposure to contaminated dust, dried dung etc due to aborted materials. Water sources, such as wells, may also be contaminated by recently aborted animals or by run-off of rain water from contaminated areas. Certain occupations such as farmers, farm labourers, animal attendants, stockmen, shepherds, sheep shearers, goatherds, pig keepers, veterinarians and inseminators are at risk through direct contact with infected animals or through exposure to a heavily contaminated environment. Persons involved in the processing of animal products such as slaughtermen, butchers, meat packers, collectors of fetal calf serum, processors of hides, skins and wool, renderers and dairy workers may be at high risk of exposure to brucellosis (Corbel, 2006). Economic impact Most data and evidence available on the economic burden of brucellosis and the benefits of its control are from high- and middle-income countries. However, it is fairly estimated that the burden of brucellosis is greatest in low-income countries (McDermott et al., 2013). Economic impacts vary depending on the main livestock species, management systems, and on the capacity of the country’s veterinary and medical systems. In developing nations, brucellosis is endemic and neglected, with large disease burdens in animals and people (McDermott and Arimi, 2002). Schwabe (1984) assessed the impact of the disease on national economy due to brucellosis in cattle and buffalo and estimated that the losses are equivalent to more than half % of total value of all meat and milk products produced in the country. The annual loss due to human brucellosis was estimated to be 30 million man-days. Kunen (1994) estimated that the losses due to brucellosis cost India at least Rs 350 million annually in terms of food animals and man-days of labour lost. A study in Tamil Nadu estimated the annual economic loss at Rs. 1180 and Rs. 2121.82 per infected sheep and goat, respectively (Sulima and Venkataraman, 2010). Roth et al. (2003) reported an average benefit-cost ratio of 3.2 for society in a scenario of 52 percent reduction from mass vaccination of livestock. Singh et al. (2015) reported that brucellosis in Indian livestock is responsible for a median loss of US $3.4 billion annually. The disease in cattle and buffalo accounted for 95.6% of the total losses occurring due to brucellosis in livestock population. The disease is responsible for a loss of US $6.8 per cattle, US $18.2 per buffalo, US $0.7 per sheep, US $0.5 per goat and US $0.6 per pig. These losses are additional to the economic and social consequences of the disease in humans. Constraints in the control of Brucellosis 1. Increased trade movement of animals and commercial dairy farming is spreading the disease across the states easily. 85 

 

2.

There is no policy for slaughter of infected animals or compensations.

3.

Lack of public awareness on economic importance of the disease, management of infected animals, zoonotic implications and lack of access to timely diagnosis are other factors precipitating the survival and spread of the disease.

Prevention and control Brucellosis has been eradicated from the island of Malta 90 years after the discovery of the disease (Wyatt, 2009). Unfortunately, this has not been the fate of other areas around the world where the disease has been endemic for thousands of years and still continues to thrive and spread. Countries that have controlled brucellosis systematically have used reliable smooth live vaccines, consistent immunization protocols, adequate diagnostic tests, broad vaccination coverage and sustained removal of the infected animals. Wildlife may become intermediate or amplifier hosts from which Brucella spp. can spill over to domesticated animals and humans. Animal brucellosis is dependent on two main principles: prevention of exposure of animals to infection, and elimination of infected animals from the herd. The former can be achieved by preventing free grazing and movement along with frequent mixing of flocks of sheep and goats, control of unrestricted trade and use of local cattle yards and fairs for trading, use of semen from Brucella-free bulls for artificial insemination and maintenance of good farm hygiene. In cattle, RBPT is used as screen test followed by testing positive sera with CFT or ELISA for confirmation. The milk ring test could be used for identifying infected dairy herd with good results followed by sero-testing individual animals. The positive reactors must be segregated to check the further spread of disease in the herd. In India “test and segregation” in conjunction with vaccination is perhaps the only method which is practical and feasible (Shome et al., 2012). However, it is not feasible if incidence >2% and is not applicable in small ruminants. Extensive vaccination with approved vaccine is a better tool for the prevention of brucellosis with mass immunization the only way to bring down the incidence of brucellosis in areas with high prevalence. For cattle, B. abortus S19, a live attenuated vaccine is administered at the age of 3 to 6 months (in certain cases up to 8 months). When used routinely to attain coverage of ≥80% of population, there is a gradual decline in incidence leading to herd immunity. Where eradication is the aim, vaccination should be stopped once the incidence falls below 0.2% and the infected animals must be eliminated. The epidemiology and planning for prevention and control of brucellosis in sheep and goats are similar to that of cattle with minor adjustments. A live attenuated Brucella vaccine based on a smooth variant of B melitensis Rev-1 appears to be highly effective and is widely used to vaccinate small ruminants in parts of the world where B. melitensis is enzootic. It should be stressed that the serum agglutination test (SAT) is generally regarded as being unsatisfactory for the purposes of international trade. The complement fixation test (CFT) is diagnostically more specific than the SAT. The

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diagnostic performance characteristics of some enzyme-linked immunosorbent assays (ELISAs) and the fluorescence polarisation assay (FPA) are comparable with or better than that of the CFT, and as they are technically simpler to perform and more robust, their use may be preferred. The performances of several of these tests have been compared. For the control of brucellosis at the national or local level, the buffered Brucella antigen tests (BBATs), i.e. the Rose Bengal test (RBT) and the buffered plate agglutination test (BPAT), as well as the ELISA and the FPA, are suitable screening tests. Positive reactions should be retested using a suitable confirmatory and/or complementary strategy. General hygiene principles like thorough cleaning of all instruments used in reproductive procedures during use in between animals and prompt disinfection of area in contact with aborted materials should be followed. Surveillance approaches for Brucellosis in India ¾ A systematic screening of all animals in randomly selected herds within an identified geographical area is to be initiated. This approach gives the actual picture of prevalence in the herd and generates a baseline data on prevalence. ¾

Epidemiological investigations to be carried out to trace sources of animals which have been added to or sold from infected herds. This approach helps in arresting infection outbreak early and to pinpoint exact source of infection.

¾

Testing of animals at slaughter or markets or abortion investigations to be carried out promptly. Bulk milk ring tests are to be used widely to determine the prevalence of brucellosis in dairy cattle herds and to locate possible additional infected herds.

¾

Males in bull stations are to be periodically monitored (Shome et al., 2012).

National Control Programme on Brucellosis The NCPB was started in August, 2010 under the aegis of Department of Animal Husbandry Dairying and Fisheries (DADF) under Ministry of Agriculture, Government of India. Under this scheme the individual infected animals are not recognized and rather the village is treated as a herd with the involvement of the village milk co-operatives in diagnosis and control through vaccination. Periodical surveillance is carried out by using milk ring test for pooled milk and ELISA for random or herd screening. Calf hood vaccination is carried out using B. abortus S19 vaccination for all the female calves of 4 to 8 months in infected villages. Calfhood vaccine prevents abortions in the herds and no booster vaccination is required. The herd immunity is built in 3-5 years period and the anamnestic response helps animals to act as indicator system and prevents abortions. The programs assures very high and sustained cost benefit ratio to the farmer and dairy industry and helps to establish accredited herds/villages. The control of infection in animals reduces the human infection burden with increased awareness.

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Network programme on Brucellosis The Department of Biotechnology, Ministry of Science and Technology, Government of India has envisaged and initiated a Network Project on Brucellosis with the aim of studying the epidemiological status of Brucella infections in India, to develop novel diagnostics and vaccines and also the national repository. Research activities with the aim to further add to the existing knowledge on epidemiology is being carried out in a few institutions across the country. A joint ‘ICMR-ICAR task force on Brucellosis’ was constituted in 2012 to formulate strategies on the way forward in brucellosis epidemiology and control in India. Future prospects Even though there have been only few systematic studies to work out the situation of brucellosis in livestock population of the country, there have been unequivocal epidemiological, serological and bacteriological evidences indicating its prevalence in various species of animals in almost all the states of India. The progress made in the diagnosis and control of brucellosis is significant but a lot needs to be carried forward. A wide variety of tests are already available along with some effective vaccines. The necessary technical know-how for the formulation and execution of control and eradication program under different socio-cultural and epidemiological situations are to be in place. However, the available vaccines are far from ideal and vaccine for all species is not available. In areas where brucellosis is enzootic, good animal husbandry is a critical component to prevent transmission both among animals and between animals and humans. The risk factors such as management practices, population dynamics and biological features largely influence the epidemiology of Brucella spp. The prevalence of the infection in domestic animals and veterinarians, calls for public health education to the target groups, along with better understanding of the risk factors, better management practices such as bio-safety, prompt diagnostic services and multisectoral collaboration amongst the medical professionals and veterinarians. It is time that the misuse of vaccines is checked and suitable diagnostic tests and management procedures are incorporated at all levels of animal husbandry practices to control this scourge. References Anonymous. 1986. Joint FAO/WHO expert committee on brucellosis. VIth Report. WHO TRS No. 740, WHO, Geneva. Bandyopadhyay S, Sasmal D, Dutta T K, Ghosh M K, Sarkar M, Sasmal N K and Bhattacharya M. 2009. Seroprevalence of brucellosis in yaks (Poephagus grunniens) in India and evaluation of protective immunity to S19 vaccine. Tropical Animal Health Production, 41:587-592. Batra H V, Chand P, Ganju L, Mukherjee R and Sadana J R. 1989. Dot-enzyme linked immunosorbent assay for the detection of antibodies in bovine brucellosis. Research in Veterinary Sciences, 46: 143-46 Benkirane A. 2006. Ovine and caprine brucellosis: world distribution and control/eradication strategies in West Asia/ North Africa region. Small Rum. Res., 62 (1-2), 19-25.

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Boral, R., Singh, M., and Singh, D. K. 2009. Status and strategies for control of brucellosis: A review. Indian Journal of Animal Sciences, 79(12): 1191-1199. Chatterjee A, Mondol P, De B N and Sen G P. 1995. Cultural isolation of Brucella in relation to serum agglutinin level. Indian Veterinary Journal, 72:211-15. Corbel, M. J. (2006). Brucellosis in humans and animals. World Health Organization. Godfroid, J., Garin Bastuji, B., Saegerman, C. and Blasco Martínez, J. M. 2013. Brucellosis in terrestrial wildlife. Hemashettar B M, Patil C S, Jaikumar K, Devraj M and Nagalotimath S J. 1987. Isolation of Brucella melitensis biotype I from a cow and two of its attendents. Indian Veterinary Journal, 64: 822-25. Kumar R and Rao C V N. 1980. Porcine brucellosis in Tamil Nadu. Indian Veterinary Journal, 57: 1-4. Kunen A V. 1994. Brucellosis In: Infectious diseases, diagnosis and management in clinical practice. CBS Publishers, New Delhi. 448-49. Maansi. 1999. Sero-epidemiology of brucellosis in animals and man in Tarai region. M. V. Sc. Thesis submitted to G. B. Pant Univ. Agril. Technol., Pantnagar. McDermott J.J. and Arimi S.M. (2002). Brucellosis in subSaharan Africa: epidemiology, control and economic impact. Vet. Microbiol., 90 (1-4), 111-134. McDermott, J., Grace, D., and Zinsstag, J. 2013. Economics of brucellosis impact and control in low-income countries. Revue scientifique et technique (International Office of Epizootics), 32(1), 249-261. Panjarathinum R and Gulrajani T S. 1974. Studies on brucellosis. II. Isolation of Brucella organism in milk of cows, sheep and goats. Indian Journal of Animal Health, 13:29-35. Paulsen I T, Seshadri R, Nelson K E, Eisen J A, Heidelberg J F, Read T D, et al., 2002. The Brucella suis genome reveals fundamental similarities between animal and plant pathogens and symbionts. Proceedings of National Academy of Sciences U S A, 99:13148-53. Pillai M T, Nendunchelliyan S and Raghvan N. 1991. Serological and bacteriological detection of Brucella canis infection of dogs in Madras. Indian Veterinary Journal, 68:399-401. Polding J B. 1947. Brucellosis in India. Indian Journal of Veterinary Sciences, 147-55. Radostits, O. M., Gay, C. C., Blood D. C. and Hinchcliff, K. W. 2000. Veterinary medicine, 9th Ed., ELBS Bailliere Tindall, London, UK, pp. 870-871. Rajkhowa S, Rahman H, Rajkhowa and Bujarbaruah K M. 2005. Seroprevalence of brucellosis in mithuns (Bos frontalis) in India. Preventive Veterinary Medicine, 69: 145-51. Renukaradhya G J, Isloor S and Rajasekhar M. 2002. Epidemiology, zoonotic aspects, vaccination and control/eradication of brucellosis in India. Veterinary Microbiology, 90:183-95. Roth F., Zinsstag J., Orkhon D., Chimed-Ochir G., Hutton G., Cosivi O., Carrin G. and Otte J. (2003). Human health benefits from livestock vaccination for brucellosis: case study. Bull. WHO, 81 (12), 867-876. Sen G P and Sharma G L. 1972. Speciation of seventy eight Indian strains of Brucella, an epidemiological study. Indian Journal of Animal Sciences, 45:537. Sharma M, Batta M K, Asrani R K, Katoch R C, Joshi V B and Nagal K B. 1995. Brucella melitensis abortions among organized sheep farms in north-west states in India. Indian Journal of Animal Sciences, 65:874-75. Shome R, Shome BR, Deivanai M, Desai GS, Patil SS, Bhure SK and Prabhudas K (2006). Seroprevalence of brucellosis in small ruminants. Indian Journal of Comparative Microbiology, Immunology and Infectious Diseases, 27(1): 13-15. Shome R., Nagalingam M. and Rahman, H. 2012. National Control Program on Brucellosis: Aims and strategies. PD_ADMAS/Tech.Bull/15/2012

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Shwabe, C.W. 1984. In: Veterinary Medicine and Human Health. Waverly Press Inc. Mount Royal Guilford Avenues, Baltimore, M. D. 21202. US. 16-39 Singh, B. B., Dhand, N.K. and Gill, J.P.S. 2015. Economic losses occurring due to brucellosis in Indian livestock populations. Preventive veterinary medicine, 119(3), 211-215. Sulima, M. and Venkataraman, K.S. 2010. Economic losses associated with brucellosis of sheep and goats in Tamil Nadu. Tamil Nadu J. vet. Anim. Sci., 6, 191-192 Wanke, M M. 2004. Canine brucellosis. Animal Reproduction Science, 82-83:195-207. Wyatt, H.V. 2009. Brucellosis and Maltese goats in the Mediterranean. J. Maltese Hist. 1, 4-18. Robinson, A. 2003. Animal Production and Health Paper No. 156. Guidelines for coordinated human and animal brucellosis surveillance. Rome, Italy: FAO, 2003.

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Medicinal plants: emerging strategies for gut health, immunity and its development in poultry Jai Sunder, Sujatha Tamilvanan and Anandamoy Kundu Division of Animal Science, ICAR-Central Island Agricultural Research Institute, Port Blair, A & N Islands 744105, India

Introduction Livestock are an important and integral component of the agricultural production system in developing countries during the recent years. Antibiotics are indiscriminately used in livestock and poultry production and the phenomenon of the problem of multidrug resistance has evolved with a great concern for an organic livestock production system. The importance of traditional and alternative medicine out of medicinal plants has been realised for sustainable livestock production by the veterinary practitioners and medicinal plants have comprised a significant proportion of veterinary research. Due to isolated spread of 572 islands in remote locations of Andaman and Nicobar Islands, rural farmers are dependent on indigenous knowledge for the treatment of their livestock and poultry rather than immediate Animal Husbandry and veterinary services. A&N Islands are the hotspot of huge diversity of medicinal plants. Research works are being carried out to explore medicinal plants as growth promoter and immunomodulator in poultry. Some of the important medicinal plants have been documented in A&N Islands to treat various ailments of poultry namely; Momordica charantia (Karela, Bitter melon, papailla, bitter gourd) to treat diarrhea ; Piper longum (Long pepper, Pipli, Lendipippal) used in loss of appetite, pneumatic pains and chronic bronchitis. This paper deals with the medicinal plants of importance in rural poultry production in A&N Islands and that have been explored as enhancers for gut health and its development, immunity and growth. 1.

Enhancers of gut health

a.

Morinda citrifolia (Noni)

Morinda citrifolia L. is commonly known as Noni, belongs to the family Rubiacee (Nelson, 2006). It grows widely throughout the coastal regions of many countries including the Andaman & Nicobar group of Islands. In these islands it is commonly known as Lorang, Burmaphal, Pongee phal and Surangi by the tribal. Morinda citrifolia has a rich history in India, where it has been used as Ayurveda medicine. However, many animals do not consume the product and avoid contact with its fruit and seeds due to its strong smell and taste. The residents of South Pacific islands have noted health benefits for themselves and their animals by ingesting the Morinda citrifolia fruit (Whistler, 1985). Some animals such as pigs consume the fruit in its natural state (Fugh-Berman, 2003). Most animals have difficulty in consuming and digesting whole fruit. Over the year’s research on use of

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fruit and leaf extract, literatures are creaming upon anthelmintic (Morton, 1992), anti-inflammatory (Fletcher et al., 2013), antitumor (Jinhua et al., 2013) and hepatic stimulatory properties (Sunder et al., 2013a). Sunder et al. (2012) have studied the wide spectrum antibacterial and antifungal activity of various parts of the Morinda citrifolia extracts. They have found that the methanol, ethanol, ethyl acetate, chloroform, acetone extracts of leaf, stem bark, fruit and seed showed broad spectrum antibacterial and antifungal activity in- vitro. Extensive research work and review has been completed on utilization of Morinda citrifolia as growth promoter, immunomodulator and gut health enhancers in poultry feed (Sunder et al., 2016a). The use of lactic acid bacteria in livestock poultry feed as probiotic has been well studied by many workers as feed supplements for growth; enhance production performance and immune response (Salarmoini and Fooladi, 2011). This wonder Morinda has been scientifically proven to have synergistic effect in combination with lactobacillus sps. The compound terpenoids and phenolic compounds such as acubin, alizarin, acopoletin and other anthraquinones present in the fruit extract being responsible for the antimicrobial activity (Narimani-Rad et al., 2011) significantly reduced gut coliform load (Sunder et al., 2012 & 2015) as it was also reported by Salarmoini and Fooladi (2011). The lactobacillus metaboilizes the feed to produce lactic acid which in turn lower the pH of gut intestinal flora and inhibit the pathogenic organisms mainly the Salmonella and E. coli. Further, supplementation of Morinda juice supported the bird to convert feed efficiently so that its supplementation saved 10 percent of feed and in turn feed cost of 5.39 per bird could be saved. The juice of Morinda fruit has potency to accelerate gut development. There were significant microscopical changes in crypt depth and villi height at the level of duodenum which is the primary site for the development of immune response and where nutrient uptake takes place (Sunder et al., 2014a). The intestinal mucous villi and crypt depth of lactobacillus fed broilers were of significantly very high approximately 439.64 µ and 51.39 µ, respectively with the base having ridges. The Morinda juice improves the histological indexes of the intestinal mucosa helping the function of gut. Feeding of Morinda citrifolia fruit juice (10 ml), Kalmegh (3 g/bird/day) and Lactobacillus acidophilus (1x108cfu/ml) significantly improved the villi height and crypt depth in broiler duodenum. b. In-ovo effect of Andrographis paniculata Nees (Kalmegh) on post hatch gut development Kalmegh (Andrographis panniculata) is a promising medicinal plant commonly used in humans as an immune system booster. Main bioactive compounds are andrographolide and diterpenoid lactone. It’s immunomodulatory and growth promoting activity has been scientifically validated (Mathivanan and Kalaiarasi, 2007). Structure of intestinal mucosa can reflect the health condition of intestine (Xu et al., 2003). Significant microscopical changes also have taken place in crypt depth and villi height at the level of duodenum in the gut of progeny from breeders fed with kalmegh feed additive (Sujatha et al., 2015). The control group mucosa

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contained villi with a height of approximately 269.28±18.48 µm that was statistically (P