Indian Journal of Comparative Microbiology

Vol. 37 Number 2 July-December 2016

Print ISSN 0970-9320 Online ISSN 0974-0147

Indian Journal of Comparative Microbiology, Immunology and Infectious Diseases

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Indian Association of Veterinary Microbiologists, Immunologists and Specialists in Infectious Diseases Website: www.iavmi.org

INDIAN JOURNAL OF COMPARATIVE MICROBIOLOGY, IMMUNOLOGY AND INFECTIOUS DISEASES

The Official Organ of the

Indian Association of Veterinary Microbiologists, Immunologists And Specialists in Infectious Diseases (IAVMI)

Executive Members

Office Bearers Patron

Prof. P. K. Uppal Former Managing Director Department of Biotechnology Ministry of Science, Govt. of India H.No. 770, Sector 17A Gurgaon -122 001 Haryana Email : [email protected]

President

Prof. M. P. Yadav Ex-Director-cum-Vice Chancellor I.V.R.I. Izatnagar Ex-Vice Chancellor SVPUAT, Meerut H.No. 365, Sector-45 Gurgaon-122 003 Haryana Email: [email protected]

Vice Presidents

Dr. Ashok Kumar ADG (Animal Health) ICAR, Krishi Bhawan New Delhi -110001 E-mail : [email protected]

Dr. R. K. Singh Director-cum-Vice Chancellor ICAR-IVRI Izatnagar, Bareilly – 243122, Uttar Pradesh E-mail: [email protected] Dr. H. S. Sandha Director Department of Animal Husbandry& Veterinary Services Govt. of Punjab, 17 Bay’s Building, Chandigarh, Punjab E-mail: [email protected] ; [email protected] Dr. B. Pattnaik Director ICAR-Directorate of Foot and Mouth Disease IVRI Campus, Mukteshwar-263138, Uttarakhand E-mail: [email protected] Dr. S. M. ByreGowda Director Institute of Animal Health and Veterinary Biologicals Bengaluru-560024 E-mail: [email protected] Dr. S. K. Jand Principal Ram Tirath Road, Amritsar-143002, Punjab Email: [email protected] Dr. A. B. Pandey Head, Division of Virology & Station Incharge ICAR-IVRI Regional Station, Mukteshwar-263138, Uttarakhand Email: [email protected] Dr. S. K. Das Professor & HOD, Microbiology COVSc, Khanapara, Guwahati, Assam E-mail: [email protected] Dr. Rishendra Verma Emeritus Scientist ICAR-IVRI, Izatnagar, Bareilly-243122, Uttar Pradesh E-mail: [email protected] Dr. S. Kilari Head, Research & Development Off. Pune-Nagar Road, Wagholi, Pune – 412 207 Maharashtra E-mail: [email protected] Dr. Ashis Paturkar Prof.& Head and Associate Dean Veterinary College, Parel, Mumbai Email: [email protected] Dr. G. Ravi Kumar Professor, Zoonoses Research Laboratory Centre for Animal Health Studies, TANUVAS Madhavaram Milk Colony, Chennai - 600 051, Tamil Nadu E-mail: [email protected] Dr. D. K. Singh Principal Scientist Veterinary Public Health Division ICAR-IVRI, Izatnagar, Bareilly-243122, Uttar Pradesh E-mail: [email protected]

Dr. V. P. Singh Director, National Institute of High Security Animal Diseases Anand Nagar, Bhopal – 462 022 Madhya Pradesh E-mail : [email protected] General Secretary

Dr. A. K. Tiwari Head, Division of Biological Standardization Deemed University ICAR-IVRI Izatnagar – 243122 Bareilly, Uttar Pradesh E-mail: [email protected]

Joint Secretary cum Treasurer

Dr. Y. P. S. Malik National Fellow Div. of Biological Standardization ICAR-I.V.R.I., Izatnagar – 243 122 Bareilly, Uttar Pradesh Email: [email protected]

Editor-in-Chief

Dr. Divakar Hemadri Principal Scientist ICAR-NIVEDI Post Box No. 6450 Yelahanka, Bengaluru-560064 Email : [email protected]

Indexed/Abstract with:

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NAAS Rating 2017 - 4.49 CNKI Scholar

 

EBSCO Discovery Indian Science

 

Summon(ProQuest)  Google Scholar DRJI  ISRA-JIF  J-Gate

INDIAN JOURNAL OF COMPARATIVE MICROBIOLOGY, IMMUNOLOGY AND INFECTIOUS DISEASES

The Official Organ of the Indian Association of Veterinary Microbiologists, Immunologists And Specialists in Infectious Diseases (IAVMI)

EDITORIAL BOARD Editor-ln-Chief

Dr. Divakar Hemadri Principal Scientist ICAR-NIVEDI, PB No. 6450, Yelahanka, Bengaluru-560064 Email: [email protected], Phone:080-23093100

Editors

Dr. Minakshi Prasad Professor and Head, Animal Biotechnology LUVAS, Hisar- 125001, Haryana. Email: [email protected] Dr. Satish B. Shivachandra Senior Scientist ICAR-NIVEDI, PB No. 6450, Yelahanka, Bengaluru-560064 Email: [email protected] Dr. (Ms.) Jyoti B. Dutta Prof. of Epidemiology & Preventive Medicine College of Veterinary Sciences AAU, Khanapara Campus, Guwahati-781022, Assam E-mail : [email protected] Dr. Pallab Chaudhuri Principal Scientist Bacteriology & Mycology, ICAR-IVRI, Izatnagar, Bareilly-243122, Uttar Pradesh Email: [email protected] Dr. Mayank Rawat Principal Scientist Division of Biological Standardisation, ICAR-IVRI, Izatnagar, Bareilly-243122, Uttar Pradesh Email: [email protected] Dr. P. K. Patil Senior Scientist Division of Aquatic Animal Health and Environment, ICAR-CIBA #75, Santhome High Road, Raja Annamalai Puram, Chennai- 600028, Tamil Nadu Email: [email protected] Dr. S. S. Patil Senior Scientist ICAR_NIVEDI, PB No. 6450, Yelahanka, Bengaluru-560064 Email: [email protected]

Indian Journal of Comparative Microbiology, Immunology and Infectious Diseases, an official organ of the Indian Association of Veterinary Microbiologists, Immunologists and Specialists in Infectious Diseases (IAVMI) publishes the articles of original research work pertaining to the Microbiology, Immunology and Infectious diseases of livestock, poultry and fish. For details visit: http://www.indianjournals.com/ ijor.aspx?target=ijor:ijcmiid&type=home Contact Editorial Office for any assistance related to online submission Editorial Office: ICAR-National Institute of Veterinary Epidemiology and Disease Informatics (ICAR-NIVEDI), PB No. 6450, Yelahanka, Bengaluru-560064 Phone: 08023093100, Email:[email protected] Email: [email protected]/[email protected] Any queries related to submitted article may be sent to: Dr. Divakar Hemadri, Editor-in-Chief, IJCMID, Email: [email protected]

Editorial Dear Readers and contributors As we are approaching the dawn of New Year- 2017, it is time to make new resolutions and targets for the days ahead. With your support and continuous patronage so far we have been able to bring out the issues of the journal in time. It has been the policy of the Indian Association of Veterinary Microbiologists, Immunologists and Specialists in Infectious Diseases(IAVMI) to maintain quality of the research publication since inception. It is my pleasure to inform you that the journal’s NAAS rating has improved from 3.5 to 4.49. However, it is not the time to be complacent, rather should resolve and focus to achieve the coveted NAAS rating of 6.0. I believe, this is a pragmatic achievable target with every one’s whole hearted support and continued patronage. You all know that we are publishing two issues in a year and many of you might have felt that we should increase the number of issues per year. As a part of enhancing Journal’s visibility and reader’s interest, starting from the year 2018, we are planning to bring out a special issue every year. The topic for the special issue will be “Emerging diseases of pigs in India”. The announcements will be in the journal’s website very shortly, which would give a specialized focused research area/forum for researcher’s working in the realm of emerging diseases of pigs in India to compile their findings and contribute in the form of research/ review articles to reach out greater number of readers across India.Again, your whole hearted support and contributions are highly appreciated. Wishing you all A Very Happy New Year-2017.

(Divakar Hemadri) Editor-in-Chief

Indian Journal of Comparative Microbiology, Immunology and Infectious Diseases CONTENTS Vol. 37

No. 2

July-December 2016

Review Article 1.

Biofilm – an eternal chronicle of bacteria Sadhana Singh Sagar, Rajesh Kumar and Shilpa Deshpande Kaistha

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Mini Review 2.

Bacterial biofilms as oral vaccine candidates in aquaculture T.N. Vinay, S.K. Girisha, Roshan D’souza, Myung-Hwa Jung, T.G. Choudhury and S.S. Patil

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Research Articles 3.

Seroprevalence of Mycoplasma gallisepticum in different parts of India Surajit Baksi, Bhumika F. Savaliya, Bhargavi Trivedi and Nirav Rao

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4.

Identification and in-silico analysis of hepcidin from Tor putitora (Hamilton) Preeti Chaturvedi, Meenakshi Dhanik, Veena Pande and Amit Pande

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5.

Phenotypic characterization of Staphylococcus Spp and PCR-based identification of Staphylococcus aureus isolated from subclinical mastitis of cows B.B. Bhanderi and M.K. Jhala

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6.

Pathogenicity and genotyping of fowl adenoviruses associated with Gizzard erosion in commercial layer grower chicken in India N.R. Bulbule, V.V. Deshmukh, S.D. Raut, C.D. Meshram and M.M. Chawak

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7.

Characterisation of antibiogram and some virulence attributes in methicillin resistant Staphylococcus aureus (MRSA) of canine origin Ritika Yadav, Vinod Kumar Singh, Amit Kumar and Sharad Kumar Yadav

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8.

Comparative evaluation of indirect-ELISA and IS900 PCR assay for diagnosis of ovine Johne’s disease S.Y. Mukartal, D. Rathnamma, H.D. Narayanaswamy, S. Isloor, S.S. Patil, S.S. Manjunatha, C. Prakash, K. Sarvesha, C.B. Ramesha and M.E. Anuradha

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9.

Growth kinetics and protein expressions in biofilms of Streptococcus agalactiae isolated from bovine mastitis cases M. Nasim Sohai, S. Isloor, D. Rathnamma and S. Sundareshan

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Contents

Printed & Published by: Diva Enterprises Pvt. Ltd. on behalf of Indian Association of Veterinary Microbiologists, Immunologists and Specialists in Infectious Diseases (IAVMI), Hisar, Printed at: Spectrum, B-122/3A, Inside Ambedkar Gate, Jagat Puri, Delhi110051, Published at: Diva Enterprises Pvt. Ltd., B-9, A-Block, L.S.C., Naraina Vihar, New Delhi 110028, India, Editor-in-Chief: Dr. Divakar Hemadri

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Review Article

Indian J. Comp. Microbiol. Immunol. Infect. Dis. Vol. 37 No. 2 (July-Dec), 2016: 45-56

BIOFILM – AN ETERNAL CHRONICLE OF BACTERIA Sadhana Singh Sagar1*, Rajesh Kumar1 and Shilpa Deshpande Kaistha2 Associate Professor, Department of Environmental Microbiology, School for Environmental Sciences, Babasaheb Bhimrao Ambedkar University (A Central University), Lucknow-226025, Uttar Pradesh, India 2 Assistant Professor, Department of Microbiology, Institute of Bioscience and Biotechnology, Chhatrapati Sahu Ji Maharaj University, Kanpur-208024, Uttar Pradesh, India 1

ABSTRACT Biofilm is a complex community of microorganisms in which microbes are embedded in a matrix of exopolymeric substances (EPS). EPS of biofilm render three-dimensional organisation and provides protection against environmental stresses. Biofilms formed on abiotic as well as on biotic surfaces lead to acute or chronic infections. Biofilm-associated infections are very fiddly to control by antimicrobials because of knotty organisation ofbiofilm. The better consideration about the biofilm organisation lends a hand to find out novel techniques to control over them. In this review, we have illustrated the complexity ofbiofilm as well as its regulatory mechanism and control mechanisms. Keywords: Biofilm, Signalling molecule, Bacteriophage, Quorum sensing, Bacteria Received: 4 December, 2016 Accepted: 15 December, 2016 DOI No.: 10.5958/0974-0147.2016.00010.6

INTRODUCTION Planktonic growth of microbes rarely exists in nature (Costerton, 1999). More than 99% of bacteria exist in nature in the form of biofilm (Costerton et al., 1987). Biofilm is defined as an assembly of microbial cells that are irreversibly associated with a surface (either inert or living) and enclosed in a matrix of exopolymeric substances (EPS). The term ‘biofilm’ was coined by Costerton (1978). Since then, it has been well documented that biofilmcoupled microbes are physiologically distinct from planktonic cells (Donlan, 2002; Hall-Stoodley et al., 2004). Bacteria can build up biofilm onabiotic as well as on the biotic surfaces (Donlan, 2001; Flemming and Wingender, 2001). Biofilm of medical implants is a serious cause of concern for the medical practitioners. According to Donlan (2002), biofilm-associated cells are 1000 times more resistant to antimicrobials than planktonic cells (Donlan, 2002). Thus, biofilm-coupled infections are tricky to eradicate with commercially available antimicrobial. WHY BIOFILM IS SO COMPLICATED A biofilm is composed of microbial cells and EPS. EPS of biofilm contain polysaccharides, uronic acids, proteins, nucleic acids, lipids, phospholipids and humic *Corresponding author E-mail: [email protected]

Indian J. Comp. Microbiol. Immunol. Infect. Dis.

substances (Jahn and Nielsenx, 1998; Wingender et al., 1999; Sutherland, 2001). According to Tsuneda et al. (2003), proteins and polysaccharides account for 75–89% of the biofilm EPS composition, indicating that they are the major components. eDNA is extracellular DNA, which is degraded DNA of dead bacterial cells. Bacterial population in biofilms is accompanied with extracellular DNA (eDNA; Dubnau, 1999; Lorenz et al., 1991) and it appears that eDNA may serve as a cell-to-cell interconnecting compound in many biofilms. However, exopolymers, outer membrane proteins and a variety of cell appendages such as pili and flagella may also function as a part of the biofilm matrix. Components of the biofilm matrix are usually produced by the bacteria itself. That is why biofilm formation in natural and industrial environment allows bacteria to develop resistance to amoebae, chemically diverse biocides, host immune clearance and antibiotics (Costerton, 1999). STEPS OF BIOFILM FORMATION Biofilm formation carried out by following steps as shown in Fig. 1. i. ii. iii. iv.

Attachment, Formation of microcolonies, Formation of matrix, Formation of macrocolony (maturation of biofilm)

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5) Dispersed of Biofilm cells

Dispersal of biofilm mediated by secondary messenger molecule released by mature by biofilm

1) Attachment

2) 1-Formation of Microcolony

Adherence of bacteria to a surface is mediated by its surface appendages like, flagella, pili, curli and OMPs

3) Formation of matrix

Motility factor inhibited after formation of microcolony consequence of inhibition of swimming motility

4) Mature biofilm

EPS and Quorum sensing molecule help in maturation of biofilm

Fig. 1: Steps of biofilm formation

Attachment to the Surface Initial stage of biofilm formation is the attachment of planktonic cells to a surface. This process of attachment is reversible and dynamic. During this stage, bacteria may rejoin or detached from the surface (Banin et al., 2005; Wu and Outten, 2009). This process is facilitated by repulsive and weak interaction forces. Eventually, the attachment of cells to surface gets irreversible. In some cases, pili are involved in attaining the irreversible attachment to the surface. In, Pseudomonas aeruginosa type ÀV, pili facilitate the irreversible attachment (O’Toole and Kolter, 1987; Klausen et al., 2003). Formation of Matrix and Microcolonies EPS: The EPS provide strength and hydrodynamic environment to the biofilm. EPS of biofilm are very complex in nature and it is composed of humic substances, polysaccharides, lipids, proteins and nucleic acids as given in previous section. It imparts the three-dimensional structure to the biofilm as well as formed matrix around the colony. EPS of biofilm are act as diffusion barrier, which prevents the penetration of any type of harmful substance inside the biofilm (Donlon, 2002).

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Quorum sensing: Bacterial biofilm is just like house. Like in a house, people use different kinds of language to communicate with each other, in the same manner bacteria use quorum sensing for communication. Quorum sensing is a density-based mechanism, in which, bacteria interact with each other through the chemical signals. Quorum sensing plays a very significant role in formation of the microcolony. Quorum sensing is vital component for the formation of biofilm. At high density, microbes secrete autoinducer molecules in the extracellular environments, which turn on the quorum sensing system (Kjelleberg and Molin, 2002). Activation of quorum sensing circuit occurs, when the concentration of autoinducer exceeds beyond the threshold (Figure 2). The luxI/luxR system is a prototype of a quorum-sensing system used by many Gram-negative bacteria. The quorum-sensing system is quite different in Gram-positive and Gram-negative bacteria; detail about the quorum-sensing system is discussed in the next paragraph. Quorum-sensing system was first elucidated in the luminescent marine bacterium Vibrio fischeri, in that organism this system regulates light production (Fuqua et al., 2001; Losick and Kaiser, 1997). Bioluminescence in such bacterium is achieved when the cells accumulate at threshold level. lux gene in V. fischeri controls the

Vol. 37 No. 2 (July-Dec), 2016

Biofilm – An eternal chronicle of bacteria

1) QSmolecule

QSmolecule

Planktonic cells Biofilm

2) Regulator 4) Expression of gene 3) Promoter

Quorum sensing molecules

Structural genes

Promoter

Quorum sensing operon

Fig. 2: Quorum sensing mechanism in bacteria

expression of bioluminescence (Henke and Bassler, 2004; Kaplan and Greenberg, 1985). lux gene is responsible for the production of autoinducer, which synthesises N-(3 oxohexanoyl)- L -homoserine lactone. Binding of autoinducer to the N-terminal regulatory domain enhances the multimer formation by inducing LuxR at the C-terminal domain to activate transcription from both Lux operons (Koch et al., 2005). All luminescent bacteria utilise long fatty acid aldehyde for the luminescence and this reaction is catalysed by luciferase enzyme and fatty acid reductase complex (encoded by LuxCDE). Hybrid quorum-sensing system: Vibrio harveyi has hybrid quorum-sensing systems that comprise the prototype of both Gram-positive and Gram-negative bacteria (Bassler et al., 1993; Bassler, 2002). Hybrid quorum-sensing system causes bioluminescence in V. harveyi (Bassler et al., 1994), type III secretion in Escherichia coli (Day and Mauralli, 2001), production of virulence factor (vir B) in Shigella (Day and Mauralli, 2001). Recently, it has been proposed that dental bacteria used this system for biofilm formation (Li et al., 2002; McNab et al., 2003). This system comprises acyl homoserine lactone (AHL)-type autoinducer. In addition to the AHL-type autoinducer, an interspecies autoinducer called AI-2, a furonosyl borate


ester, has also been identified as a signal for hybrid quorumsensing system. Quorum-sensing system in Gram-positive bacteria: Gram-negative bacteria use AHL molecules as autoinducer, while Gram-positive bacteria use secretary proteins as an autoinducer molecule for activation of quorum-sensing system. Different bacterial species such as Bacillus subtilis, Staphylococcus aureus and Streptococcus pneumoniae employ quorum-sensing system to regulate phenotypic expression (Novick, 2003). When the density of microbial cells reaches a threshold level, then the secreted autoinducing peptide (AIP) signal is detected by the two component sensor kinase. This activates the response regulators, which facilitate DNA binding and hence alters the transcription of target genes involved in quorum sensing (Novick et al., 1995). AIP-deficient mutants form more robust biofilms than the wild-type strain (Vuong et al., 2004; Yarwood et al., 2004), leading to the conclusion that the AIP quorum-sensing system negatively regulates biofilm formation. As has been observed in other organisms, biofilm formation in S. aureus is highly dependent on the culture medium. When S. aureus is cultured in the presence of glucose, agr gene expression

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is repressed (Regassa et al., 1992). This may be partially responsible for the observation that glucose can promote S. aureus biofilm formation (Boles and Horswill, 2008). Quorum sensing in Gram-negative bacteria: In Gramnegative bacteria, AHL type of quorum-sensing system is present and it is involved in direct interaction with plants and animals. AHL is synthesised by enzymes, which are member of LuxI family of acyl-HSL synthases (Fuqua et al., 1994; Schaefer et al., 1996; Parsek et al., 1999; More et al., 1996). Different LuxI homologs generate different acyl-HSLs. In P. aeruginosa, RhlI primarily catalyses the synthesis of N-butyryl-HSL (C4-HSL), and LasI directs the synthesis of N-(3-oxododecanoyl)-HSL (3OC12-HSL). AHL signalling is critical for virulence of the plant pathogen Erwinia carotovora (Pirhonnen et al., 1993) and for virulence of P. aeruginosa in mouse modelsof lung (Tang et al., 1996), in burn infections (Rumbaugh et al., 1999), in invertebrates (Tan et al., 1999) and in plants (Rahme et al., 1995). P. aeruginosa contains two types of quorum-sensing systems LasR-I and RhlR-I. LasR is a transcriptional regulator that responds primarily to LasI-generated signal and 3OC12-HSL, and RhlR is a transcriptional regulator that responds best to the RhlI-generated signal, C4-HSL (Parsek and Greenberg, 2001). RhlR responds best to the RhlI-generated C4-HSL. RhlR then activates expression of genes required for production of a variety of secondary metabolites such as hydrogen cyanide and pyocyanin (Pesci and Iglewski, 1997). Quorum sensing plays a very significant role in biofilm formation in P. aeruginosa. Mutation in LasI leads to dramatic change in biofilm formation. LasI mutant’s biofilm is arrested after microcolony formation, while before maturation of the microcolonies, it is converted into thick structured assemblages. Quorum-sensing mechanism operates in specific environmental niches, where the appropriate environmental signals are available. However, conditions such as the nutritional status of the environment and even the presence of conjugative plasmids can lead to quorum-sensing cues being bypassed or overridden (Ghigo, 2001; Collet et al., 2007; Gonzalez et al., 2006; Rice et al., 2005; Reisner et al., 2006). Presumably, all of these signals feed into intricate cellular signalling networks which respond under the appropriate prevailing conditions. Hence, the biofilm forming regulatory mechanism through the quorum-

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sensing system can be identified by the elucidation of all of these networks (Schaefer et al., 2001). Signalling inside biofilm at molecular level C-di-GMP: Cyclic-di-GMP is a secondary messenger molecule, which controls biofilm formation as well as virulence factors of a bacterial cell. Cyclic-di-GMP was first discovered as a regulator of cellulose synthesis in Glucoacetobacter xylinus, and it emerged as a major bacterial secondary messenger (Huang et al., 2003; Romling et al., 2005; Ross et al., 1990; Tamayo et al., 2007). The intracellular concentration of cyclic-di-GMP is controlled by the GGDEF domain proteins with diguanylate cyclase activity and the EAL domain proteins with cyclic-di-GMP-specific phosphodiesterase activity (Romling et al., 2005). GGDEF domains catalyse the condensation of two molecules of GTP to generate cyclicdi-GMP, while the EAL domains catalyse the hydrolysis of cyclic-di-GMP to generate the dinucleotide 5-pGpG. EAL domain-containing proteins, including a large number of proteins that contain both the EAL and GGDEF domains, regulate virulence gene transcription, biofilm formation, motility and adhesion which have been reported in various pathogenic bacteria. Several proteins in the human pathogen Vibrio cholerae, including VieA and CdgC, have been implicated in biofilm formation, motility and virulence factor production (Tischler and Camilli, 2004; Karatan et al., 2005; Jenal and Malone, 2006; Lim et al., 2006). An EAL domain protein was found to control lateral flagellar-gene expression and swarming behaviour in Vibrio parahaemolyticus (Kim and McCarter, 2007). In Salmonella enterica, the disruption of the EAL domain protein CdgR weakens bacterial resistance to hydrogen peroxide and accelerates bacterial killing by macrophages (Hisert et al., 2005). In the opportunistic pathogen, P. aeruginosa, the EAL domain-containing protein FimX controls switching motility and biofilm formation (Huang et al., 2003; Kazmierczak et al., 2006) and the BifA protein controls biofilm formation and swarming (Kuchma et al., 2007). A systematic analysis of the GGDEF and EAL domain proteins in P. aeruginosa identified several other EAL domain proteins as being involved in virulence expression and biofilm formation (Kulasakara et al., 2006; Kazmierczak et al., 2006). Because of regulation of virulence characteristic of a microorganism by EAL domain proteins, their study makes them potential targets for developing antibacterial agents that aim to neutralise virulence functions. Vol. 37 No. 2 (July-Dec), 2016


MATURATION Microcolony is converted into mature form by multiplication and secretion of polymeric substances. Quorum sensing also plays a very important role in the maturation of biofilm. Klausen et al. (2003) showed nonmotile subpopulation of P. aeruginosa which formed stalks, while the motile subpopulation formed mushroom-shaped caps on these stalks by migration with type IV pili. Boles et al. (2004) reported functional diversification of P. aeruginosa cells in the mature biofilm; a subpopulation of cells form wrinkly colony, while another forming a mini colony. The matured biofilm is characterised by a complex architecture (Wood et al., 2010; Davey et al., 2003; Bridier et al., 2011). DISPERSAL OF BIOFILM There are several factors which are responsible for the dispersal of biofilm such as oxygen, nutrient depletion condition and presence of toxins (Sauer et al., 2004; Karatan and Watnick, 2009; Hong et al., 2010; Rowe et al., 2010). Biofilm can be dispersed in the form of bulk, clumps and in individual form from the attached surface. Nutrient depletion can also be a cause of dispersal in Pseudomonas species (Hunt et al., 2004; Gjermansen et al., 2005). Secondary messenger molecule cyclic-di-GMP plays a pivotal role in the formation of biofilm, whereas the reduced level of these molecule switches the sessile form to planktonic form (Morgan et al., 2006; Pruss et al., 2006; Barraud et al., 2009;Wood et al., 2010; Chua et al., 2015). Bacteriophage-associated enzyme can also dissolved the matrix of biofilm which eventually lead to dispersal of biofilm (Hanlon et al., 2001; Meng et al., 2011). SIGNIFICANCE OF BIOFILM Persistent infections are a global problem for medical practitioner, and it is reported that biofilm plays a significant role in persistent infections. Bacterial infections that affect internal organs are cystic fibrosisa chronic lung infection disease caused by P. aeruginosa (Singh et al., 2000), E. coli causes urinary-tract infections (Anderson et al., 2004) and Mycobacterium tuberculosis causes human tuberculosis (Ojha et al., 2008). A spectrum of indwelling medical devices or other devices used in the healthcare has been shown to harbour biofilms, consequently assessable rates of device-associated infections (Donlan, 2001; Costerton et al., 2003). The role of biofilms in the


contamination of medical implants has been well established. Electron microscopy studies have revealed the sign of presence of biofilm on the medical implants (Gristina et al., 1988; Nickel et al., 1989; Neu et al., 1994; Busscher et al., 1998). It is evident that bacterial biofilms on prosthetic valves are the leading cause of endocarditis in patients who have undergone heart valve replacement. Amongst patients who develop these infections have near about 70% mortality rate (Hyde et al., 1998). Millions of catheters (central line, intravenous and urinary catheters) used in the medical field are potential source of infection and ideal surfaces for formation of biofilm. Biofilm formation can also occur on contact lenses, and these biofilms are thought to contribute to keratitis (Elder et al., 1995; Gorlin et al., 1996; McLaughlin-Borlace et al., 1998). Another example of a likely biofilm-mediated infection is chronic ear infection (otitis media). These infections are often caused by biofilm bacteria (Dingman et al., 1998). Thus, biofilms are the damaging cause of huge medical and social resources every year. RESISTANCE MECHANISM OF BIOFILM AGAINST ANTIMICROBIAL COMPOUNDS The bacterial cells enclosed within the biofilm matrix are extremely resistant to antibiotic treatments. Such resistance can be explained hypothetically given by (Fig. 3). First, the EPS of biofilm act as a physical/chemical barrier, thus preventing many antimicrobials (Costerton et al., 1995; Lewis, 2001; O’Toole et al., 2000; Thien and O’Toole, 2001). Moreover, EPS are negatively charged due to eDNA and function as an ion-exchange resin, which is capable of binding a large number of the antibiotic molecules with positive charge that are attempting to reach the embedded biofilm cells. Second, embedded biofilm bacteria are generally not divided rapidly, virtually antibiotics only act on rapidly dividing cells (Brown et al., 1988; Wentland et al., 1996; Wingender et al., 1999). Slow growth rate inactivates the RelA protein, which regulate synthesis of ppGpp. In bacterial cells, ppGpp inhibits the anabolic processes as well as suppress the activity of autolysin, SLT, which make cells resistant for antibiotic and autolysin, respectively (Cashel et al., 1996; Betzner et al., 1990; Prakash et al., 2003). 49

Singh et al.

1- Exopolysachharides as a physical as well as chemical barrier 8- Quorum sensing system contribute in antibiotic resistance

2-Slow growth rate of biofilm cells

7- Inactive growth rate of persister cells provide resistance from antibiotic attack in the biofilm

6- Acceleration in drug resistant plasmid multiplication

3-Antibiotic degrading enzymes secreted by biofilm cells as well as EPS of biofilm

4- Alteration in cell wall composition of biofilm cells

Bacterial Biofilm

5- Reactive intermediate made by the biofilm which deactivate antibiotics Fig. 3: Biofilm resistance mechanisms

Third, resistant mechanism, EPS of biofilm matrix contain the  lactamase enzyme in the immobilised form, which degraded  lactame antibiotics. Biofilm of P. aeruginosa contains 32 times more  lactamase enzyme than the planktonic cells (Tuomanen et al., 1986). Fourth, up to 40% of the cell-wall protein composition of bacteria in biofilms is altered from planktonic cells (O’Toole et al., 2000; Potera, 1999). The membranes of biofilm bacteria might be better equipped to pump out antibiotics to protect themselves from the antimicrobials. Fifth, biofilm matrix deactivated antimicrobials faster than its diffusion, for example reactive oxidants such as hypochlorite and hydrogen peroxide (De Beer et al., 1994; Chen and Stewart, 1996; Xu et al., 1996; Thien and O’Toole, 2001). These antimicrobial oxidants are products of the oxidative burst of phagocytic cells and poor penetration of these may partially account for the inability of phagocytic cells to destroy biofilm microorganisms. Sixth, biofilms also provide an ideal environment for the exchange of extra chromosomal DNA (Plasmid) responsible for antibiotic resistance, virulence factors and survival capabilities at accelerated rates, all these things make it prefect milieu for emergence of drug resistance pathogens (Donlan, 2002; Ghigo, 2001; Hausner and Wuertz, 1999).

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Seventh, formation of persister cells inside the biofilm provides the resistance against antibiotics (Lewis, 2010). Persister cells are characterised as non-growing cells; hence, the antibiotics which target only the actively dividing cells are not effective (Jayaraman, 2008). They are extremely resistant for the antibiotics as well as host immune response (Lewis, 2008). Eighth, quorum-sensing system in bacterial biofilm contributes in antibiotic tolerance and resistance to the host immune system (Lewis, 2008). Characteristics of biofilm that can be important in infectious disease processes include a) Detachment of cells or biofilm aggregates may result in bloodstream or urinary-tract infections or in the production of emboli (Raad et al., 1992). b) Increased resistance to antimicrobial compounds and immune system: Cells may additionally exchange resistance plasmids within biofilm leading to exaltation of virulence factors, and/or antibiotic resistance (Sedor and Mulholland, 1999; Donlan, 2000; Murga et al., 2001). c) Production of toxins: Gram-negative bacteria within the biofilm produce endotoxins (Donlan, 2002). Vol. 37 No. 2 (July-Dec), 2016


Endotoxins of such bacteria are dangerous to patients undergoing hemodialysis, which can be transported in the dialysis membrane of dialyser through dialysate (Vincent et al., 1989). Measurement of level of endotoxin in the hemodailysis tube indicates a correlation with bacterial colonisation (Holland et al., 2000). Endotoxin release from the biofilm increased the biocompatibility of dialysis fluid and leads to chronic inflammatory complications in patients (Marion-Ferey et al., 2005). d) Quorum sensing systems contribute in expression of multiple virulence factors in microorganism (Lewis, 2008). STRATEGIES USED FOR THE CONTROL OF BIOFILM Bacterial biofilm is very difficult to control or eradicate from surfaces; therefore, for the removal of biofilm, alternative approaches rather than conventional antibiotics have been proposed. There are a range of small molecular compounds, which inhibit the biofilm formation of Gram-positive as well as Gram negative-bacteria. Opperman et al. (2009) screened a number of molecules and found that aryl rhodanines inhibit the early stage of biofilm formation in Gram-positive and Gram-negative bacteria. Sambanthamoorthy et al. (2011) reported that benzimidazole inhibits the biofilm formation by inhibition of second messenger molecule C-di-GMP. Cis-2-decenoic acid disperse the biofilm in many bacteria was identified by Davies and Marques (2009). Brominated furanones, natural compound isolated from the marine algae Delisea pulchra, has very good antibiofilm efficacy against S. aureus, P. aeruginosa, Enterococcus feacalis, Streptococcus mutan and Staphylococcus epidermidis, which is isolated from rhizospheric bacterium Stenotrophomonas maltophilia BJ01 (Singh et al., 2013; Khan and Husain, 2002). Many heavy metals have also antimicrobial activity and can also inhibit biofilm formation for example silver ion inhibits the biofilm formation; but biofilm matrix proteins degrade silver ions. Application of silver nanoparticles for the control of bacterial biofilm is a good approach. As nanoparticles have large surface area due to their large surface area, they can easily kill the biofilm cells (Stevens et al., 2009; Ansari et al., 2015).


Thuptimdang et al. (2015) reported that biofilm is more susceptible for nanoparticle when EPS is stripped from it. Silver nanoparticles play very important role in the control of biofilm formation of P. aeruginosa and S. epidermidis. Mohanty et al. (2012) reported control of biofilm of P. aeruginosa with starch-mediated synthesised silver nanoparticles. Habash et al. (2014) reported control of biofilm of P. aeruginosa PA01 biofilm with citrate-capped nanoparticles. Gum Arabic cappedsilver nanoparticle controls biofilm formation of clinical isolates P. aeruginosa in concentration-dependent manner. Furanones is chemical compound isolated from the red alga D. pulchra; it inhibits the biofilm formation. Coating of such biofilm inhibiter can limit the biofilm formation in the medical implants. Dispersin B, produced by a Gram-negative Actinobacillus actinomycetemcomitans, disperses the biofilm by targeting the matrix of biofilm (Kaplan et al., 2004). In addition of these chemical compounds, there are some biological agents, which can limit the biofilm formation. These include bacteriophage and their associated enzyme. Two strategies employed by bacteriophage in fighting biofilm are blocking of biofilm development and eradication and removal of existing biofilm cells (Rózalska et al., 2010). The most advantageous feature of bacteriophage is that they are host-specific self-replicating entities; once administered, they self-replicate in the host and destroy them (Parasion et al., 2014). Moreover, bacteriophage cocktails can remove mixed biofilms of bacteria (Lu and Collins, 2007; Lehman and Donlan, 2015). Use of bacteriophage is more economical, ecological and eco-friendly. CONCLUSION Significant studies done by the previous researchers on biofilm reveal the complexity of the biofilm organisation. Moreover, the complexity of biofilm makes it highly dynamic and versatile so that it can survive in any type of stress environment. There are lots of techniques available for the control of biofilm, but all of them are unable to completely inhibit biofilm formation. Researchers are continuously reporting about controlling of biofilm, but the fact is that control of biofilm is still problematic. Discovery of novel signalling molecules and biological strategies such as use of bacteriophages can control bacterial biofilm to some extent. If we talk about the phage therapy, bacteria have evolved resistance mechanism

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Mini Review


BACTERIAL BIOFILMS AS ORAL VACCINE CANDIDATES IN AQUACULTURE T.N. Vinay1*, S.K. Girisha2, Roshan D’souza3, Myung-Hwa Jung4, T.G. Choudhury5 and S.S. Patil6 Scientist, School of Molecular Diagnostics and Prophylactics, ICAR-Indian Instt. of Agril. Biotechnology, Ranchi, Jharkhand 2 Assistant Professor, Department of Fisheries Microbiology, College of Fisheries, Mangalore, Karnataka 3 Research Professor, Research Institute of Bacterial Resistance, Yonsei Univ. College of Medicine, Seoul, Republic of Korea 4 Postdoctoral Fellow, Department of Aqualife Medicine, Chonnam National University, Yeosu, South Korea 5 Assistant Professor, Dept. of Aquatic Health and Environment, College of Fisheries, Central Agril. Univ. (Imphal), Tripura 6 Senior Scientist, ICAR-National Institute of Veterinary Epidemiology and Disease Informatics, Bangalore, Karnataka 1

ABSTRACT The application of new and novel strategies to develop effective vaccines is essential in aquaculture health management. The bacterial biofilm-based oral vaccine development is an innovative technique. Biofilm-based vaccines have very good gastrointestinal stability in comparison with vaccines from conventional free cells (whole bacterial cell) of bacteria. Biofilms are produced by growing bacteria in a nutrient depleted condition providing a substrate for its attachment. The biofilm thus produced, inactivated and delivered orally to various fish species have demonstrated good humoral and protective response than with free cell-based vaccines. The studies have been reported from herbivore carps, omnivore catfish, and carnivore murrel. This review provides the overview of recent advances in bacterial biofilm-based oral vaccine development for aquaculture species. Keywords: Bacterial biofilm, Biofilm vaccine, Oral vaccine, Fish vaccine, Aquaculture Received: 5 December, 2016 Accepted: 15 December, 2016 DOI No.: 10.5958/0974-0147.2016.00011.8

INTRODUCTION Aquaculture systems are highly threatened by various bacterial diseases, and vaccination of fish helps to control the spread of diseases. Vaccines are an important part of healthy and sustainable aquaculture (Brudeseth et al., 2013). Oral vaccines targeting gut mucosal immunity are best suited for aquaculture species as the strategy is very practical for mass vaccination compared with injection vaccination, which is laborious and leads to handling stress in fish (Munang’andu et al., 2015a; Carmen and Forlenza, 2016). Mucosal immunity plays a major role in protecting fish from various pathogens as the mucosal surfaces like skin, gills and gut are the first line of defence in aquatic species (Rombout et al., 2014; Koshio, 2016). There are several innate components which act as preliminary barriers for pathogens. However, adaptive immune response is the one which is important in vaccine development. The major hurdle in oral vaccine development is the degradation of antigens in *Corresponding author E-mail: [email protected]


gastrointestinal tract before reaching the immune cells (Munang’andu et al., 2015b; Carmen and Forlenza, 2016). Several vaccine approaches have been developed and evaluated for oral delivery, which includes feeding free cells of inactivated pathogens, recombinant proteins, live attenuated pathogens, DNA-based vaccines and these antigens encapsulated with various micro and nanoparticles (Carmen and Forlenza, 2016). The demand for oral/ mucosal vaccines in aquaculture is ever increasing due to the obvious advantages and the practicality. Among several approaches, the use of bacterial biofilms as vaccine candidates is an important approach in developing oral vaccines, as most of the bacteria are capable of biofilm formation (Jacques et al., 2010). The use of bacterial biofilms as vaccine candidates for aquaculture species dates back to the late 1990s. Bacterial biofilms are well-structured, multicellular communities, capable of adhering and growing on various biological and inert surfaces (Prakash et al., 2003; Jacques et al., 2010; Wu et al., 2014; Gupta et al., 2016). They encase in self-produced extra cellular (glycocalax) matrix

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called extra polymeric substances which are resistant to several agents (Jacques et al., 2010; Gupta et al., 2016). Though, this nature of bacterial adaptation is a severe threat in various bacterial diseases, this very nature can be exploited to develop oral vaccines for fish in aquaculture. Biofilms of most of the bacteria can be formed artificially and used for experimental purposes. As mentioned earlier, the biofilm of a bacteria consists of a cellular matrix resistant to various agents, which has the ability to survive the harsh gastric conditions, and protect the antigens from degrading before reaching the immune-responsive sites, which is an essential factor in developing oral vaccines (Mutoloki et al., 2015). Furthermore, it has shown that the bacterial biofilms express several other proteins than in its regular form, which might also add to the increased antigenicity (Asha et al., 2004). Several advances have been made in developing biofilm-based oral vaccines for fish. This review aims to explain the principle of bacterial biofilm formation, and its application as vaccine candidates for fish with the best proofs of the concept. FORMATION OF BACTERIAL BIOFILMS Basically, biofilm is formed as a survival strategy in an unfavourable condition to the bacteria, like nutrient depletion or other environmental stressors (Prakash et al., 2003). Formation of bacterial biofilm involves several steps, where it first attaches to a substrate and then forms micro colonies before forming matured three-dimensional structures and detaches from the substrate (Prakash et al., 2003; Jacques et al., 2010). Further, it depends on the ability of bacteria to communicate with other cells via quorum sensing by releasing different chemical signals (Gupta et al., 2016). As an example for biofilm-based vaccine development for aquaculture species, the gramnegative Aeromonas hydrophila, a secondary pathogen of fish, which causes huge mortality and economic losses in aquaculture, has been demonstrated for biofilm formation (Azad et al., 1997, 1999, 2000). The bacteria were grown in a nutrient depleted condition, providing chitin flakes as an inert substrate, with continuous stirring of 4–6 h a day for 4 days developed biofilm of A. hydrophila, and the biofilm was harvested and inactivated to nullify its pathogenicity and retain its antigenicity and used as oral vaccine candidates by mixing in the feed for fish in different studies. Fig. 1 provides the schematic representation of bacterial biofilm formation and application as an oral vaccine candidate. 58

GUT MUCOSAL IMMUNITY AND ORAL VACCINES FOR FISH Mucosal immune system of fish consists of innate and adaptive systems, which facilitate in developing immune memory to mucosal vaccines (Koshio, 2016). All the three immunoglobulin types (IgM, IgT/IgZ and IgD) characterised in fish are detected in the mucosal surface of fish (Hansen et al., 2005; Hu et al., 2010; Ballesteros et al., 2013). In addition, several studies have also reported the presence of cellular immunity in fish vaccinated via mucosal route (Kumari et al., 2013; Munang’andu et al., 2013; Chettri et al., 2014; Kai et al., 2014; Rombout et al., 2014), and Munang’andu et al.(2015a) have reviewed in detail. The innate and adaptive immune system of fish mucosal surfaces has been extensively reviewed recently by Rombout et al. (2011, 2014); Gomez et al. (2013); Lazado and Caipang (2014), which provides an excellent knowledge on the mucosal immunity of fish. Munang’andu et al. (2015a) have reviewed the mechanism of mucosal vaccines of fish, providing insights for mucosal vaccine development. The first oral/mucosal vaccine for fish was developed in 1942 by Duff against Aeromonas salmonicida, which gave a very encouraging result protecting significantly more fish than in unvaccinated groups with high antibody titre (Duff, 1942). This vaccination strategy was neglected, when the use of antibiotics and other chemicals became more predominant in aquaculture health management. However, the realisation and experience of ill effects of using chemotherapy and antibiotics have led to the reemergence of vaccine research, and due to the practicality, mucosal/oral vaccines are the most preferred vaccines and have become a major research area in several laboratories globally. With many existing approaches, using bacterial biofilms as mucosal vaccines have made a considerable progress and has been demonstrated in different species of fish, with and without proper stomach and has provided very good protection along with the hint of inducing systemic immunity in fish. BACTERIAL BIOFILMS AS FISH ORAL VACCINES The idea of using bacterial biofilms for oral vaccines of fish to resist the gastric enzymes in the gut and to provide immune memory against the pathogen infection was conceptualised by Dr. K.M. Shankar and executed by Azad et al. (1997) for the first time for fish vaccines against A. hydrophila.


Bacterial biofilms as oral vaccine candidates

Bacteria Bacteria Substrate

Grow bacteria in nutrient depleted conditions providing a substrate

Produce self-encasein in Produceand and self-encase extracellular polymeric extracellular polymeric substance (EPS) substance (EPS)

Substrate

Bacteria attaches to substrate and starts forming biofilm

Substrate

Bacterial biofilm based oral vaccine development for fish Vortex to detach bacterial biofilm from substrate Orally immunize fish through feeding Inactivate biofilm Mix inactivated bacterial biofilm with fish feed Fig. 1: Attachment and growth of bacterial biofilm on the substrate and its application as oral vaccine candidate for aquaculture species

A. hydrophila isa major fish pathogen of great importance to aquaculture industry. It leads to motile aeromonad septicaemia, and the clinical signs are marked by swelling of tissues, dropsy, red sores, necrosis, ulceration and haemorrhagic septicaemia, and it infects several aquaculture species (Karunasagar et al., 1989). Extensive studies have been performed with A. hydrophila-based biofilm as potential vaccine candidates. The data is available from fish of all kinds of feeding habits, that is, herbivores carps, omnivore catfish and carnivore murrel (Azad et al., 1997, 1999, 2000; Nayak et al., 2004; Vinay et al., 2013; Siriyappagouder et al., 2014). Gastrointestinal stability of the biofilm-based oral vaccines was reported by employing monoclonal antibodybased immunofluorescence and immunoperoxidase assays (Azad et al., 2000). As expected, the biofilm-based vaccine antigens were detected in the lumen of hind gut even after 48 h of administration in comparison with free cells (planktonic form of bacteria), which formed the basis and provided the much needed foundation for the biofilmbased oral vaccine development research (Azad et al., Indian J. Comp. Microbiol. Immunol. Infect. Dis.

2000). The presence of large amount of biofilm antigens in lumen indicates the resistance of antigen to gastric enzymes and available in required quantity for the development of proper immune memory (Azad et al., 2000), which otherwise may induce immune tolerance due to lower antigen doses after antigen destruction in gastrointestinal tract (Munang’andu et al., 2015b). The protective response to the bacterial challenge has been assessed by intraperitoneal bacterial challenge model in all the studies post-oral administration of biofilm and free cell-based vaccines, and a significant protection has been observed in all the reported studies, which indicates that the mucosal vaccination can protect fish from pathogen infection. Table 1 provides the relative percentage survival details of all the studies. Humoral response to support the protective response of vaccination was assessed through serum agglutination assay in initial studies which provided valuable information. However, accurate measurement of specific immunoglobulin (IgM) levels in vaccinated fish is important to evaluate the efficacy of vaccines (Morrison and Nowak, 59

Vinay et al. Table 1: Protective response of bacterial biofilm oral vaccines Species Pathogen RPS in Vaccinated Groups Biofilm Free Cell Catla catla A. hydrophila 60 20 Labeo rohita A. hydrophila 67, 83 50, 25 Cyprinus carpio A. hydrophila 70 60 Clarias batrachus A. hydrophila 100, 91, 100 42.1, 29, 31 Channa striatus A. hydrophila 88 30 RPS; relative percentage survival.

2002). In continuation, we developed a monoclonal antibody-based ELISA and measured the specific antibody titre post-oral vaccination of biofilm-based vaccine to Labeo rohita (Vinay et al., 2013). Siriyappagouder et al. (2014) too reported the employment of ELISA to measure specific antibody titre and reported the humoral response to oral vaccination of biofilm-based vaccine in Channa striatus. These studies demonstrate that the mucosal immunisation induces specific IgM in vaccinated fish. CONSTRAINTS IN ORAL VACCINE STRATEGY Oral vaccination is no doubt an efficient and practical approach to target mass vaccination in aquaculture as it is very laborious to inject every fish. But the approach is not devoid of constraints. Some of the important constraints of oral vaccine strategy in aquaculture are as follows: 1. Equal distribution of vaccine containing diet to every fish cannot be monitored. 2. Antigen intake cannot be monitored to quantify the efficacy. 3. Universal oral vaccine cannot be designed against a pathogen to different species as the gut enzymes vary based on the feeding habit of the fish. 4. Large quantity of antigen needs to be produced. 5. Lower dose or very high dosing may lead to immune tolerance. FUTURE PROSPECTS Biofilm-based vaccines or immunostimulant development is an interesting prospect for the control of particular disease in aquatic animals. The development of biofilm-based vaccine and its efficacy is already demonstrated, and it can also be further used to develop effective immunostimulants. In the absence of immune memory in shrimps, they need a continuous stimulation

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Reference Azad et al. (1997, 1999) Azad et al. (1997, 1999), Vinay et al. (2013) Azad et al. (1997, 1999) Nayak et al. (2004) Siriyappagouder et al. (2014)

with immunostimulants to maintain the immune response and show resistance against various infections (Sung et al., 1994). Several immunostimulatory agents including killed bacteria, peptidoglycans, glucans and lipopolysaccharides have been used to increase the resistance of shrimps (Smith et al., 2003). Bacterial biofilms are excellent immunostimulating agents as they express several other antigenic proteins and are also resistant to gastric enzymes. A study has demonstrated the use of biofilm of Vibrio alginolyticus administered orally to Penaeus monodon showed an increased innate immune response with protective response against V. alginolyticus and WSSV challenge indicating the efficacy and nonspecific cross protection (Sharma et al., 2010a, 2010b). This is an interesting prospect for the development of immunostimulants for shrimp and other aquatic organisms. Further, the concept can be applied to develop biofilmbased vaccines for other major bacterial pathogens prominent in aquaculture industry. CONCLUSION It is clear that bacterial biofilm-based vaccine development is a novel approach to develop oral vaccines for aquaculture species. The main advantage of biofilmbased vaccine is that it can be used for oral vaccination without the need for an encapsulation and additional adjuvant for immune stimulation. The present studies are mainly restricted to A. hydrophila. However, the aquaculture system is plagued with several bacterial diseases, and all bacteria are capable of biofilm formation. This novel concept can be applied to develop more biofilmbased oral vaccines for fish in future. ACKNOWLEDGEMENT This study was a part of the project title ‘Biotechnological Interventions in Fish Health management (IIAB-FHM-01-01)’, funded by the Indian Council of Agricultural Research, India.


Bacterial biofilms as oral vaccine candidates

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Vinay et al. Sharma, K.S.R., Shankar, K.M., Sathyanarayana, M.L., Sahoo, A.K, Swamy, N. and Rao, S. (2010b). Evaluation of immune response and resistance to diseases in tiger shrimp, Penaeus monodon fed with biofilm of Vibrio alginolyticus. Fish Shellfish Immunol., 29:724–732. Siriyappagouder, P., Shankar, K.M., Naveen, K.B.T., Patil, R.R. and Byadgi, O.V. (2014). Evaluation of biofilm of Aeromonas hydrophila for oral vaccination of Channa striatus. Fish Shellfish Immunol., 41:581–585. Smith, V.J., Brown, J.H. and Hauton, C. (2003). Immunostimulation in crustaceans:dose it really protect against infection?. Fish Shellfish Immunol., 15:71–90.

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Sung, H.H., Kou, G.H. and Song, Y.L. (1994). Vibriosis resistance induced by glucan treatment in tiger shrimp (Penaeus monodon). Fish Pathol., 29:11–17. Vinay, T.N., Patil, R.R., Suresh, B.P.P., Rana, R. and Shankar, K.M. (2013). Evaluation of the efficacy of Aeromonas hydrophila biofilm vaccine in Labeo rohita employing monoclonal antibody based ELISA. Open access Scientific Reports., 2:684. Wu, H., Moser, C., Wang, H.Z, Hoiby, N. and Song, Z.J. (2014). Strategies for combating bacterial biofilm infections. Int. J. Oral. Sci., 7:1–7.


Research Article


SEROPREVALENCE OF MYCOPLASMA GALLISEPTICUM IN DIFFERENT PARTS OF INDIA 1

Surajit Baksi1, Bhumika F. Savaliya2*, Bhargavi Trivedi3 and Nirav Rao4 AVP-Technical Services, 2Veterinary Officer-Animal House, 3Assistant Manager- Technical Services, 4Senior ExecutiveQuality Control, Hester Biosciences Ltd., Ahmedabad, India ABSTRACT

A serological study in broiler breeders was conducted to know the prevalence of Mycoplasma gallisepticum (MG) in different states (n = 7) of India, in a period starting from January 2015 to July 2016. A total of 1,285 sera samples were collected from the different states, and ELISA (Enzyme Linked Immuno Sorbent Assay) was conducted to know the antibodies titre against MG. Out of 1,285 sera samples, 412 were found positive to MG and overall prevalence of 32.06% was observed. Seroprevalence of MG infection was found significantly (p < 0.05) higher during winter season (58.10%) than in summer (7.43%). Further prevalence was recorded in different age groups, with significantly (p < 0.05) higher occurrence in young birds (42.75%) compared with adults (10.46%). Out of seven states, much higher prevalence (50.00%) was observed in Telangana state, whereas lower prevalence (20.00%) was noticed in Karnataka. Keywords: Chronic respiratory disease, Mycoplasma gallisepticum, ELISA, India, Seroprevalence, Avian mycoplasmosis Received: 9 September, 2016 Accepted: 28 December, 2016 DOI No.: 10.5958/0974-0147.2016.00012.X

INTRODUCTION Chronic respiratory disease (CRD/Avian mycoplasmosis) is one of the major bacterial diseases among avian diseases in India. It is caused by an important pathogen of poultry, Mycoplasma gallisepticum (MG). Primarily, CRD is a disease of chickens and turkeys, but also infects many other domestic and wild birds all over the world (Bradbury et al., 1993; Jordan and Amin, 1980). All ages of chickens and turkeys are susceptible to CRD; however, young birds are more prone to infection than adults (Nunoya et al., 1995). Mycoplasmosis is a chronic and slow-spreading contagious disease in birds characterised by obstinate hacking cough, sneezing and tracheal rales (Chakrabarti, 1993). It is not a killer disease like Newcastle or Gumboro disease, but in complicated cases, birds may die (Jordan and Amin, 1980). The economic loss incurred results from poor feed conversion ratio (FCR) of broiler, declined egg production in layer, reduced hatchability in breeder flock, down-graded broiler meat and condemnations of carcasses (Carpenter et al., 1982). The disease can transmit both horizontally and vertically and remain in the flock constantly as subclinical

form (Bencina et al., 1988). Avian mycoplasmosis may be diagnosed by different methods such as morphology of causal agents, cultural characteristics, physical, biochemical and serological properties (Ley and Yoder, 1997). Serology is the only reliable tool for detecting the subclinical infection in the flock (Prodhan, 2002). Due to economic importance, diagnosis and prophylaxis of avian mycoplasmosis have received attention. Therefore, the aim of the present study was to know the seroprevalence of mycoplasmosis in chickens so as to take an effective control measure. MATERIALS AND METHODS Sample Collection A total of 1,285 blood samples were collected from different states of India as mentioned in Table 1, to screen for the presence of antibodies to MG. At the time of blood collection, no clinical signs suggestive of CRD were observed in any of the flocks. Blood samples were collected from the jugular veins and transferred to the Hester Biosciences Ltd, Anand, for further investigation. Blood samples were centrifuged to separate the serum and then stored into -20 °C until it was tested.

*Corresponding author E-mail: [email protected]


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Competitive ELISA All the 1,285 serum samples were screened for the presence of MG antibodies using commercial ELISA kit (IDEXX Laboratories, USA). The ELISA was performed following the collection of all the blood samples as per the manufacturer’s protocol and instructions. Before use, all the samples and reagents were allowed to room temperature and homogenised by gentle mixing. All the serum samples were diluted at 1:500 with sample diluents provided by the manufacturer. A total of 100 µl negative control was added to wells A1 & B1, and 100 µl of positive control was added to wells C1 & D1. Then, 100 µl of diluted sera samples were added into the appropriate wells and incubated at 18 to 26 °C for 30 min by covering the plate with lid. The plates were washed three to five times by the 350 µl of the wash solution using ELISA washer (Nunc, Denmark). Then, 100 µl of the conjugate was added to each well and incubated at 18 to 26 °C for 30 min. Following washing, 100 µl of TMB (Tetra methyl benzidine) substrate reagent was added into appropriate each wells and incubated at 18 to 26 °C for 15 min. Finally, 100 µl of stop solution was added to each well to stop the reaction. Then, the microtitre ELISA plate was placed in the ELISA reader (BiotekELX 800) and the intensity of the colour produced from the ELISA test was measured photometrically at 650 nm wavelength. Statistical Analyses The result from serology was entered in Microsoft Excel spreadsheet (Microsoft Corp., USA) before analysing in Stata 13 software (Statacorp. LP College Station, TX, USA). The dependent variable serostatus [seropositive (1) and seronegative (0)] were binary responses and resembled a binomial distribution. For analysis purpose, birds with different ages were categorised into birds aged 0–15, 15–30, 30–45 and more than 45 weeks. A logistic model with ‘farm’ as a random effect, to adjust for clustering of birds within same farm, was used to model association, between serostatus (positive or negative) and categorical risk factors: age effect and season effect. The above logistic model was used for both univariate and multivariate analyses. RESULTS AND DISCUSSION A total of seven states (Haryana, Karnataka, Madhya Pradesh, Telangana, West Bengal, Maharashtra and Andhra Pradesh) of India were selected for the study. Out of total

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1,285 serum samples, 412 samples were found positive for MG antibodies. The overall prevalence of MG was found to be 32.06%. A slightly higher prevalence was observed by the researchers of Bangladesh: Sikder et al. (2005) reported 56.86% seropositive layer in chickens for MG infection in Patuakhali district of Bangladesh, and they also reported 58.90% seropositive layer in chickens for MG infection in some model breeder poultry farms in Feni district of Bangladesh. Other researchers also found higher prevalence of MG in poultry. Bencina et al. (1987), Godoy et al. (2001), Prodhan (2002), Biswas et al. (2003), Zhang et al. (2001), Dulali (2003) and Abdu et al. (1983) reported 56.54, 59.10, 57.15, 54.90, 53, 52 and 47.54% seroprevalence of MG infection in chickens, respectively. The findings of MG infection in the present study were in agreement with that of Biswas et al. (1992), Amin et al. (1992) and Udhayavel et al. (2016), who recorded 13– 53% seroprevalence of MG infection in some selected poultry farms in the southern part of Bangladesh and India. (Discuss the status of MG infection in Indian scenario as reported by previous studies.) The study revealed that out of seven states, higher prevalence (50.00%) of MG was found in Telangana state (195 samples were found positive out of 390), whereas much lower prevalence (20.00%) was found in Karnataka (48 samples were found positive out of 239) (Table 1). A total of four groups (0–15, 15–30, 30–45 and >45 weeks) were made to understand the effect of age on occurrence of CRD. The study showed higher prevalence (42.75 and 42.66%) of MG in age group of 30–45 and 15–30 weeks, whereas lower prevalence (8.21%) was observed in the age group of 0 to 15 weeks of age. The study also showed that age of the birds has significant (p < 0.05) effect on the prevalence of MG (Table 2). Similar Table 1: State-wise seroprevalence of avian mycoplasmosis State No. of No. of Prevalence Samples Positive (%) Samples Haryana 94 26 27.65 Karnataka 239 48 20.00 Madhya Pradesh 79 25 31.64 Telangana 390 195 50.00 Maharashtra 96 20 20.83 West Bengal 225 52 23.11 Andhra Pradesh 162 46 28.39 Total 1,285 412 32.06


Seroprevalence of Mycoplasma gallisepticum Table 2: Age-wise seroprevalence of avian mycoplasmosis Age Group No. of No. of Prevalence p Value* (Weeks) Samples Positive (%) Samples 0–15 365 30 8.21 15–30 293 125 42.66 30–45 283 121 42.75 45 344 36 10.46 Total 1,285 412 32.06 *Significant, if p < 0.05. Table 3: Season-wise seroprevalence of avian mycoplasmosis Seasons No. of No. of Prevalence p Value* Samples Positive (%) Samples Summer 242 18 7.43 Winter 253 147 58.10 0.05), but the viable count was higher than that of 1.25 per cent and 2.5 per cent of LB glucose medium. Hence, 0.625 per cent LB glucose with 0.3 per cent bentonite clay was found to be optimum for maximum biofilm production by SA, when compared with nutrientrich conditions such as 1.25 per cent and 2.5 per cent concentration (Table 3 and Fig. 2). The duration of lag and log phase was only 1 day in case of planktonic cells, whereas in biofilm cells, it was 3 days. The stationary and decline phase were extended in case of biofilm cells when compared with planktonic cells which was very much short (Fig. 3). The decline of biofilm cell population was very gradual and survived at very high counts even after 10 days of post-inoculation, whereas planktonic cells underwent rapid autolysis. This clearly indicates that the biofilm cells


survived for longer periods compared with planktonic cells in adverse conditions. Similar observations were reported with respect to Salmonella Gallinarum (Prakash and Krishnappa, 2002), E. coli (Veeregowda, 2003) and S. aureus (Naveen Kumar, 2005) who have used TSB as growth medium for biofilm formation. The number of biofilm cells colonising the inert surface increases when the surrounding medium is nutritionally restricted. The increased persistence observed in biofilm cell population could be explained by the fact that biofilm exopolysaccharide matrix traps the available nutrients and promotes the sustained growth of the biofilm microcolonies, further adsorbing and concentrating the available nutrients on to itself, thus assuring that the adherent bacteria have maximum access to nutrients (Watnick and Kolter, 2000). Solid surface provided a resting place as well as concentrating nutrients to biofilms there upon in E. coli (Zobiel and Anderosn, 1936). Optimisation of LB Glucose for Maximum Biofilm Formation by Microtitre Plate Method The biofilm growth kinetics of different bacteria in flasks using different growth media with bentonite clay as an inert surface has been studied extensively (Veeregowda, 2003; Naveen Kumar, 2005), but the drawbacks of this method are: it is very much laborious and time-consuming task, interpretation of the results is based on the viable organisms, the bacterial cells losing life during processing and detachment from biofilm matrix will be ignored. To overcome this, microtitre plate method was applied to study the growth kinetics of biofilm and free cells of SA. This method is quite easy and at the same time very accurate because the results will be calculated on the basis of absorbance of the growth covering both viable and dead cells. The microtitre plate method has been applied for growth kinetics studies of oral biofilm forming bacteria by Kirti (2011) and dental plaque forming bacteria by Sheetal (2011). In the present study, different concentrations of LB glucose (0.31, 0.625, 1.25 and 2.5 per cent) were tried to study the growth kinetics of SA on the surface of polystyrene microtitre plates as inert surface for attachment and biofilm formation. The OD values were recorded daily and plotted against time interval to determine biofilm formation by SA. The highest OD value of 1.85 was obtained on day one post-inoculation in 2.5 per cent LB glucose, whereas highest OD values of 1.07, 1.45,

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1.36 and 2.13, respectively, were obtained for 0.31, 0.625, 1.25 and 2.5 per cent LB glucose on day two postinoculation, and there was decrease in OD values after third day onwards for 0.31, 0.625, 1.25 and 2.5 per cent LB glucose. Further, the biofilm production was significantly (P < 0.001) high on day two post-inoculation in 2.5 per cent LB glucose with a mean OD value of 2.13 (Table 4, Fig. 3 & 4). Similar observations were also reported by Harmeet et al. (2009) who studied the growth kinetics of group B streptococci using TSB as growth medium with maximum biofilm cells in 48 h. During this study, it was observed that the growth kinetics in LB glucose with bentonite clay in flasks produced maximum biofilm cells on day three post-inoculation in 0.625 per cent of LB glucose compared with the growth kinetics in microtitre plates that has given maximum biofilm growth on day two post-inoculation in 2.5 per cent LB glucose. This might be because of high quantity of the medium in the culture flask containing 50 ml and less quantity of the culture medium in microtitre plates with only 180 µl of medium. The growth of the organism remains same but the quantity of the medium is very less in the plates compared with the flask. Quantification of Proteins Laboratory practice in protein purification often requires a rapid and sensitive method for the quantitation of proteins. Bradford protein assay is a simple and accurate spectroscopic/colorimetric procedure for determining the concentration of protein in solution using the Coomassie G-250 dye. The method is based on the principle of binding of Coomassie G-250 dye to proteins (Bradford, 1976). The protein content of SA grown under biofilm mode on bentonite clay and on glass beads and under free cells mode were estimated using a protein-dye-binding method according to Bradford (1976). The protein concentration obtained was 311.5 µg/ml, 270 µg/ml and 210 µg/ml, respectively, for biofilm cells on bentonite clay, glass beads and free cells, and that the protein concentration was high in biofilms grown on bentonite clay, followed by biofilm grown on glass beads and lowest in free cells. Protein Expression Profile of S. agalactiae grown in Biofilm and Free Cell Mode Bacteria grown under biofilm mode are demonstrably and profoundly different from their free cell counterparts in expression of proteins. The adhesion of bacteria to surface triggers the expression of a number of genes, 112

making the biofilm cells phenotypically different from the free cells of the same species (Costerton et al., 1995). There is a mounting evidence to show that both up and down regulations of a number of genes occur in the attaching cells upon initial interaction with the substratum. Becker et al. (2001) found that 22 per cent of the genes were up-regulated and 16 per cent were down-regulated in biofilm forming P. aeruginosa. Genes encoding for enzymes involved in glycolysis or fermentation (Phosphoglycerate mutase, Triosephosphate and Alcohol dehydrogenase) are up-regulated in biofilm forming S. aureus (Becker et al., 2001) and summarised that the upregulation of these genes could be due to oxygen limitation in the developed biofilms, favouring fermentation. In general, during biofilm formation, approximately 40 per cent of genes are altered by at least two fold (O’Toole et al., 2000). Bacteria often encounter environments where nutrient availability is limited, and they must adapt accordingly as in-vivo growth most likely occurs at submaximal rates because of nutrients limitation. In the current study, analysis of proteins extracted from the biofilm and free cells of SA was carried out by SDS-PAGE to detect any variation and/or relatedness between the cells grown under different nutritional conditions with regard to their over expression or repression. For this, SA SA3 was grown in biofilm mode using bentonite clay and glass beads as well as in free cell mode using LB glucose medium, and the proteins extracted were subjected for SDS-PAGE analysis. Several polypeptide bands ranging from 69.9 to 16.8 kDa were detected. Analysis of Protein Profiles of S. agalactiae Biofilms on Bentonite Clay and Glass Beads and Free Cells In the present study, lot of variations in the expression of proteins in biofilm and free cells of SA were observed. The protein expression profile of biofilms grown in 0.625 per cent LB glucose with 0.3 per cent bentonite clay for 3 days had differed from biofilms grown on glass beads by over expression of 47.63, 45.5, 40, 29.37, 22.85, 20.5 and 16.8 kDa proteins with expression of 24.8 kDa as unique protein. The proteins of biofilms grown on bentonite clay also differed from free cells by over expression of 47.37, 45.5, 40, 35, 29.37 and 16.8 kDa proteins with expression of 38.2, 33.17, 31.24, 24.81, 22.85 and 20.5 kDa as unique proteins (Table 5 and Fig. 5). Over expression of the proteins in biofilms over free cells has also been reported by Prakash and Krishnappa Vol. 37 No. 2 (July-Dec), 2016

Growth kinetics and protein expressions in biofilms of Streptococcus agalactiae

(2002) in S. Gallinarum, Vadakel and Krishnappa (2002) in P. multocida, Veeregowda (2003) in E. coli and Naveen Kumar (2005) in S. aureus. In the present study, interestingly, it has been observed that the expression of 24.8 kDa protein in biofilms grown in 0.625 per cent LB with one per cent glucose on bentonite clay was not the same as expressed in biofilms grown on glass beads. At the same time, the repression of 68.8 kDa protein in the biofilms grown on bentonite clay and expression in biofilms grown on glass beads was also noticed. This is in consensus with results obtained by Anwar et al. (1984), Lorian et al. (1985), Shand et al. (1985) and Costerton et al. (1987), who reported that changes on the cell surface occur in the cell of the same bacterial species in response to variation in nutrient status, surface growth and other environmental factors. Protein expression profile of biofilms grown in 2.5 per cent LB glucose with 0.5 mm glass beads for 2 days had differed from biofilms grown on bentonite clay by repression of 24.8 kDa protein, lower expression of 45.5, 38.2, 35, 29.37 and 20.5 kDa proteins and moderate expression of 47.63, 40, 22.85 and 16.8 kDa with expression of 68.8 kDa as unique protein. Further, the proteins of biofilms grown on glass beads also differed from free cells by over expression of 40 and 16.8 kDa, with expression of 38.2, 33.17, 31.24, 22.85 and 20.5 kDa as unique proteins. Our results are in comparison with those of previous studies that clearly show that electrophoresis method could possibly provide a valuable information. As a result, it is concluded that the banding pattern obtained by SDS–PAGE of the proteins of biofilm and free cells readily provide a data for the differentiation of protein expression profile of biofilm and free cells. Bacteria do not express the same antigens in-vitro and in-vivo, a mechanism known as phase variation. When the bacteria are grown in-vivo, they must cope up with a hostile environment in which certain nutrients are not abundant and where the host attempts to eliminate them in different ways. For example, under in-vivo conditions, iron and certain oligo elements are in scarce, bacteria must express adhesion factors (fimbriae, pili, adhesins etc.). These structures are lost in-vitro because in energy terms, the production of polysaccharides, adhesins, iron sequestering proteins and others are costly and unnecessary, in fact, the bacteria sometimes lose genetic information need to produce these structures and are never able to express them again. The way in which phase


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Results and Discussion

vi. Acknowledgements vii. References. Journal The list of references as per the format such as: Chomczynski, P. and Sacchi, N. (1987).Single step method of RNA isolation by acid guanidium thiocyanate- phenol– chloroform extraction. Anal. Biochem., 162: 156–159. For multiple authors format should be: Audarya, S.D., Sanyal, A., Mohapatra, J.K., Pattnaik, B. (2015). Polymearse chain reaction for amplification of IL1 gene from peripheral blood mononuclear cells of cattle.Indian J. Comp. Microbiol. Immunol. Infect. Dis., 36: 8789. Book Mohinuddin, S.M.(2007). Infectious diseases of domestic animals.1stedn.International Book Distributing Co. Lucknow. Chapter in Edited Book Prasad, G., Sreenivasulu, D., Singh, K.P., Mertens, P.P.C., Maan, S. (2009). Bluetongue in the Indian subcontinent. In: Mellor, P.P., Baylis, M and Mertens, P.P. (Eds). Bluetongue. Academic Press, London. pp 167-195. Thesis Giridharan, P. (2003). Development and evaluation of a multiplex PCR for differentiation of foot and mouth disease virus serotypes in India. MVSc Thesis, IVRI, Izatnagar, India.

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Indian Journal of Comparative Microbiology, Immunology and Infectious Diseases CONTENTS Vol. 37

No. 1

January-June 2016

Review Article 1. Infectious Bovine Rhinotracheitis (IBR): Current Status in India B.M. Chandranaik, S.S. Patil, D. Rathnamma, K.P. Suresh and G.B. Manjunatha Reddy

1

Research Articles 2. Antibiogram of Bacterial Isolates originated from Clinical Infections of Dog in Kolkata, West Bengal S. Dey, K. Batabyal, A. Mahanti, S.N. Joardar, I. Samanta and D.P. Isore

5

3. Parameters of Controlled Expression of Potentially Toxic Recombinant Proteins in E. coli with Picornaviral 3ABC as a Model M. Uma, S. Mallavarapu, K. Nagalekshmi, P.P. Rao and N.R. Hegde

9

4. Polymerase Chain Reaction for Amplification of IL-2 Gene from Peripheral Blood Mononuclear Cells of Cattle S.D. Audarya, B. Pattnaik, A. Sanyal and J.K. Mohapatra

16

5. Evaluation of Side Effects of Adjuvanted Viral Haemorrhagic Septicaemia Vaccines Following Intra-Peritoneal Administration to Olive Flounder (Paralichthys olivaceus) T. N. Vinay, C.S. Park, S.J. Jung and S.S. Patil

19

6. Polymerase Chain Reaction for Amplification of MCP-1 Gene from Peripheral Blood Mononuclear Cells of Cattle S.D. Audarya, B. Pattnaik, A. Sanyal and J.K. Mohapatra

24

7. Seroprevalence of Coronavirus in Dairy Herds of Jabalpur Region D.P. Shrivastava, M. Swamy, A. Dubey and V. Sthevaan

27

8. Use of Indirect ELISA Based on Recombinant Protein NcSAG1 for Seroprevalence of Neospora caninum Antibodies in Sheep, Ducks and Pigeons from Egypt H.M. Ibrahim

31

9. Characterization of Listeria monocytogenes from various sources by Random amplified polymorphic DNA (RAPD) and serotyping U. Singh, S. Bobade, S.Warke and D. Kalorey

37

10. Obituary

43

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