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ThesisItem Open Access IMMUNO INFORMATIC APPROACHES IN DESIGNING VACCINE AGAINST PATHOGENIC LEPTOSPIRA THROUGH PAN GENOME REVERSE VACCINOLOGY(SRI VENKATESWARA VETERINARY UNIVERSITY TIRUPATI - 517 502. (A.P.) INDIA, 2016-12) SUDHEER, P; RANIPRAMEELA, D(MAJOR); VINOD KUMAR, N; JAGADEESH BABU, AABSTRACT: Leptospirosis is a globally important zoonotic disease caused by pathogenic Leptospira and it is a disease of livestock, pet animals, wildlife and humans throughout the world. The losses are due to reproductive problems in livestock and mortality in case of humans. Current existing leptospiral vaccines are unsuccessful due to their limitations .Hence there is a need to develop a novel and efficacious vaccine to control the disease. To overcome this, reverse vaccinology is the right choice. In recent trends, it is possible to target the common vaccine candidates with genomic information of the single organism. Based on this concept the present work was planned to study the Bioinformatics approaches to identify the vaccine candidates in designing a vaccine against pathogenic Leptopsira in Bovines In the present study complete proteomes of L.borgpetersenii hardjo bovis JB 197 and L550 were screened to identify common surface exposed proteins through Rxvi language scripts and codes. Later these common surface exposed proteins were subjected to DEG analysis. 49 essential proteins were identified .Further essential proteins were subjected to non-homology analysis against both host and gut microbiota to avoid autoimmunity using NCBI-BLAST.T-helper cell epitopes were predicted from non-homologous proteins through MetaMHCII and ProPred homology search against BoLA-DRB3, and evaluated using Vaxijen server. Three dimensional structures were built for T-cell epitopes and BoLA-DRB3 using Modeller9v.15. A total of twenty five models were generated. The model with high negative DOPE score was selected and validated using PROCHECK, ProQ, and ProSA in determining protein quality. The structures of T-cell epitopes and BoLA DRB3 were prepared before docking using protein preparation wizard of Schrodinger 2015-3. Docking and free energy calculations were performed with BioLuminate Module v 2.0 of Schrödinger software suite 2015-3. The changes in structural confirmation were monitored in terms of energy plot, RMSD and RMSF during 50 ns MD simulations run time using Desmond v4.3. In Silico analysis of L.borgpetersenii JB 197 and L550 retrieved three proteins namely Ton B dependent receptor containing single epitope, ABC permease protein with three epitopes and UVr ABC protein B with single epitope. L.ballum was selected instead of L.borgpetersenii due to its non-availability of the culture during the period of the study and 99% identity on BLASTp. The nucleotide sequence corresponding to ABC permease gene containing three epitopes was retrieved, primers were designed and PCR was standardized for the amplification of ABC permease gene. PCR purified product on sequencing analysis confirmed the presence of ABC permease gene. Then, the PCR purified product was cloned in to PRSET vector using E.coli DH5α cells .The recombinant plasmid was transformed in to E.coli BL21 (DE3) cells and expression was induced by addition of 1mM IPTG. Finally recombinant protein was extracted from lysate of E.coli BL21 (DE3) cells. Recombinant protein was analysed on SDS-PAGE for characterization. The SDS-PAGE analysis yielded 20KD of expected recombinant protein on staining with commassie brilliant blueThesisItem Open Access STUDIES ON ANTIBIOTIC RESISTANCE AMONG MAJOR BOVINE MASTITIS PATHOGENS IN ANDHRA PRADESH(SRI VENKATESWARA VETERINARY UNIVERSITY TIRUPATI - 517 502. (A.P.) INDIA, 2016-12) USHARANI, K; CHAITANYA, R.K(MAJOR); SREENIVASULU, D; PADMAJA, KABSTRACT: Mastitis remains as a major problem to the dairy industry worldwide, as it affects the quality and quantity of milk production. In the present study a total of 130 milk samples were collected from clinical (101) and subclinical cases (29) of bovine mastitis from different regions of Andhra Pradesh. Isolation of causative bacteria was carried out and a total of 105 bacterial isolates were obtained. The incidence of Staphylococcus spp. (70/105, 66.66%) was found to be high, followed by Enterobacter spp. (16/105, 15.23%), E. coli (11/105, 10.47%) and Streptococcus spp. (8/105, 7.61%). Among the 70 isolates of Staphylococcus spp., 37 were identified as coagulase positive Staphylococci (CPS) and 33 were identified as coagulase negative Staphylococci (CoNS) on tube coagulase test and in coagulase gene PCR. Multiplex PCR carried out for species identification of Staphylococcal and Streptococcal isolates, confirmed all the 37 CPS isolates as S. aureus, a CoNS isolate as S. epidermidis and six isolates of Streptococci as St. agalactiae. The in vitro antibiotic sensitivity test of Staphylococcal isolates revealed high frequency of resistance to pencilin G (48 isolates, 68.57%) followed by cefoxitin (35, 50%), oxacillin (24, 34.28%), gentamicin (6, 8.57%), ciprofloxacin (5, 7.14) and ceftriaxone (2, 2.85%). Interestingly, all the isolates were found susceptible to chloramphenicol. As cefoxitin is used as surrogate for mecA mediated oxacillin / methicillin resistance, 35 (50%) isolates that showed resistance to cefoxitin were phenotypically identified as methicillin resistant, out of which 18 were MRSA and 17 were CoNS. In PCR for mecA and mecC genes that confer methicillin resistance in Staphylococci, only ten (10/70, 14.28%) Staphylococcal isolates were found to carry mecA gene. Three of them were S. aureus and the remaining seven were coagulase negative Staphylococci. The relative frequencies of MRSA and MR-CoNS were 8.1% (3/37) and 21.2% (7/33) respectively. All these mecA positive isolates were found resistant to cefoxitin which is a surrogate for mecA mediated oxacillin/ methicillin resistance. However, six of these mecA positive isolates were found susceptible to oxacillin. None of the 70 Staphylococcal isolates carried mecC gene. Phenotypic resistance was observed in three isolates of St. agalactiae (3/8, 37.5 %), but none was found to carry resistance genes tetO and ermB in PCR. Out of 27 (11 E. coli and 16 Enterobacter spp.) isolates of coliforms, 14 (14/27, 51.85%) isolates were suspected as ESBL producers as they showed resistance to any of the 3rd generation non combination cephalosporins tested in phenotypic screening test. Among these fourteen isolates, only four (4/14, 28.57%) have shown increased diameter of inhibition zones (≥ 5 mm) with the drugs in combination with ß- lactamase inhibitors over the individual drugs and hence these four isolates were phenotypically confirmed as ESBL producers. All the 27 isolates were susceptible to ertapenem and combination drugs of 3rd generation cephalosporins with ß-lactamase inhibitors i.e. ceftriaxone + tazobactem, ceftazidime + clavulanic acid and cefotaxime + clavulanic acid. Out of 27 isolates of coliforms tested for ESBL genes, six isolates (3 E. coli and 3 Enterobacter spp.) were found carry SHV gene in m PCR-I. In m PCR-II, an isolate each of E. coli and Enterobacter spp. were found to carry CTX-M-1 gene and another isolate of E. coli was found to carry both CTX-M-1 and CTX-M-2 gene. Hence these three isolates were confirmed as ESBL producers genotypically. Among the 4 phenotypically confirmed ESBL producers, ESBL genotype was confirmed only in 3 of them (2 E. coli and 1 Enterobacter spp.) with the presence of CTX-M genes. The other ESBL isolate didnot carry any of the ESBL genes. Results of the present study indicate considerably high levels of antibiotic resistance among the major bacterial species causing mastitis in cattle and buffaloes. Hence, it is imperative to go for antibiotic susceptibility testing prior to choosing an appropriate antibiotic for treatment.ThesisItem Open Access PRODUCTION, ISOLATION AND CHARACTERIZATION OF IgY ANTIBODIES TO CANINE PARVOVIRUS(SRI VENKATESWARA VETERINARY UNIVERSITY TIRUPATI - 517 502. (A.P.) INDIA, 2016-12) ATCHUTHARAM, A; SREENIVASULU, D(MAJOR); SREEDEVI, B; ESWARAPRASAD, PABSTRACT: The overall goal of this study was to develop practical, natural, and efficient antimicrobials from egg. The existence of an IgG-like molecule in avian eggs, referred to as IgY, has been well documented, and extensive research has been carried out on its characterization, production and purification. Although it is the functional equivalent of mammalian IgG, the major serum antibody found in mammals, IgY is structurally different, and has been found to exhibit several important differences when compared to mammalian antibodies, including its physico-chemical properties and immunological capabilities. Recently, considerable research has focused on the use of IgY as an alternative to mammalian antibodies in several applications, including immune therapeutic applications, especially for the oral passive immunization against various bacteria and viruses. Much research has also been carried out on the use of IgY as a replacement for IgG in various immunodiagnostic and immune affinity purification purposes. The use of IgY offers several advantages over polyclonal antibodies produced in mammals, including providing a much more hygienic, cost efficient, convenient, humane and plentiful source of antigen-specific antibodies. Chicken immunoglobulin Y (IgY) may provide a new modality in the therapy of various infectious animal diseases. This study presents evidence of its efficacy for canine parvovirus (CPV), which is a highly infectious, fatal viral disease in dogs. Initially IgY antibody production and separation was standardized using BSA as antigen. BSA 0.2 mg/ml was used to immunize three 21weeks old white leghorn chicken. Additional booster doses were administered weekly intervals up to 6 weeks following the first injection. Sera from immunized chicken were collected on 21st day to confirm the immune response to BSA. Among three BSA immunized birds, one birds did not show any response to BSA antigen. Initially BSA specific IgY antibodies were separated from eggs laid by immunized chicken using Ammonium sulphate method. The specificity of IgY antibody raised against BSA was determined by agar gel immunodiffusion test. A clear precipitation line was observed between BSA and anti BSA IgY antibody precipitated from egg yolk, which shows specificity of immune response to BSA. Anti BSA IgY antibody levels were monitored up to 120 days by indirect ELISA and found that the titres were maintained up to 120 days of immunization with the highest titre on 75th day. The present study was conducted as a preliminary step to monitor the in vitro efficacy of IgY as a passive immunotherapeutic agent to control Canine parvovirus infection in dogs. Canine parvovirus vaccine containing 103viral particles/ml was used as antigen to immunize 21 weeks old white Leghorn chicken. The eggs from immunized chicken were collected from 1st day to 135th day. Sera from immunized chicken were also collected on 21st day to confirm the immune response to canine parvovirus. Presence of antibodies against canine parvovirus was checked using hemagglutination inhibition assay (HI). The HI titres of the serum collected from immunized hens was found to be 256 HI units. Water soluble fraction was isolated from eggs collected after the immunization and estimated the protein content and the maximum protein concentration was found (35.20 ± 1.32a mg/ml) in the water soluble fraction collected from egg yolk on 75th day of post immunization. The IgY was separated from the WSF by using Ammonium sulphate method, Sodium chloride method, Sodium sulphate method and PEG method. Among the four methods used for IgY separation, Sodium chloride method and Sodium sulphate methods were found to yield high protein content (6.70 to 6.71mg/ml) compared with Ammonium sulphate method and polyethylene glycol method. IgY was purified using DEAE cellulose column chromatography. Highest protein concentration was observed in the 4th and 5th fractions. The purity of the immunoglobulin present in the 4th and 5th fractions of DEAE cellulose column elute was checked by determining the molecular weight of the protein. A single protein band showing molecular weight 180 KDa was recorded. The titre of the purified canine parvovirus specific IgY antibody was found to be 2048 HI units. Anti canine parvovirus IgY antibody levels were monitored up to 135 days by indirect ELISA. It was found that the titres were be maintained up to 135th day with peak titre (1.186) on day 75th after immunization. Stability of canine parvovirus IgY antibody was studied by exposing to different temperatures and different pH using HI assay. The HI titre gradually decreased when the temperature increased to 50ºC and above. At 25ºC and 37ºC the IgY antibody was found to be stable when exposed to 10, 20 and 30 minutes. Purified IgY antibody when subjected to pH 7 for 8 hours its HI titre was 1024. The HI titre gradually decreased when the pH decreased below 7 and also when pH increased above 7. The stability of purified anti canine parvovirus antibody was completely lost when it is exposed to pH 3 in the presence of pepsin as reflected by complete loss of HI activity. HI titre of the IgY antibody was found to be 32 to 64 when exposed to pH 4.0 and pH 5.0 respectively in the presence of pepsin. The results indicated that the activity of immunoglobulins reduced in the presence of pepsin. Hence, there is a need to protect IgY immunoglobulins against the action of pepsin in the stomach of puppies. Present study was helpful for production of desired IgY antibodies, which can be obtained in large quantities against specific pathogen (canine parvovirus).ThesisItem Open Access STUDIES ON BETA-LACTAMASE ANTIMICROBIAL RESISTANCE IN CANINE MICROBIOTA AND SCREENING OF LACTIC ACID BACTERIA FOR PROBIOTIC ACTION(SRI VENKATESWARA VETERINARY UNIVERSITY TIRUPATI - 517 502. (A.P.) INDIA, 2016-12) NOORBASHA MOHAMMAD SHARIF; SREEDEVI, B(MAJOR); CHAITANYA, R.K; SRILATHA, ChABSTRACT: Microbiota plays a central role in host health and disease. Alterations in gut microbiota can have major consequences, both beneficial and harmful, for host health. In view of this, rectal swab samples from healthy (92) and diarrhoeic (44) dogs as well as pus swabs from pyometra (15) and otitis (15) clinical cases were analyzed. Isolation and identification of canine microbiota was carried out by conventional cultural methods. Gut microbiota isolated include E. coli (65.2% incidence in healthy versus 75% in diarrhoeic dogs), Proteus spp. (61.9% vs. 65.9%), Enterobacter spp. (26% vs. 11.3%), Klebsiella spp. (26% vs. 20.4%) and Pseudomonas spp. (28.2% vs. 45.4%). Microbiota isolated from pus swabs include Staphylococcus spp. (73.3% incidence in pyometra vs. 100% in otitis), Pseudomonas spp. (80% vs. 53.3%), Proteus spp. (0% vs. 60%) and E. coli (46.6% vs. 0%). E. coli isolates were further confirmed by PCR targeting E16S gene and also sent for serotyping based on ‘O’ antigen. Extensive usage of antibiotics in canine practice may lead to development of antimicrobial resistance in dogs. In view of this, all the isolates obtained in the present study were screened for β-lactamase resistance both phenotypically and genotypically. Overall incidence of β-lactamase antimicrobial resistance in phenotypic screening test was found to be 35.8% (125/349), which includes 54 E. coli, 40 Pseudomonas, 22 Klebsiella and 9 Enterobacter species. Of these 125 isolates, resistance to cefotaxime was observed in 80.8%, ceftriaxone in 57.6%, ceftazidime in 52% and aztreonam in 26.4% of isolates. β-lactamase resistance was detected in 34.5 and 42.7% of gut microbiota isolated from healthy and diarrhoeic dogs, respectively; 46.6 and 12.5% of microbiota isolated from pyometra and otitis pus samples, respectively. All the Proteus and Staphylococcus spp. were found to be highly sensitive or intermediately sensitive to β-lactam antibiotics. Overall incidence of ESBL phenotype in phenotypic confirmatory test was found to be 14.6% (51/349), with highest incidence detected in E. coli (31%, 31/100) followed by Klebsiella (21.2%, 7/33) and Pseudomonas (19.6%, 13/66) species. ESBL phenotype was detected in 12.5 and 17.7% of gut microbiota of healthy and diarrhoeic dogs, respectively and 33.3% of microbiota of pyometra pus samples. Detection of β-lactamase genes in canine microbiota was carried out using a set of three multiplex PCR assays and a single uniplex PCR assay. The overall incidence of β- lactamase genes in canine microbiota was found to be 57.3% (200/349). Predominant β- lactamase genes detected in canine microbiota include blaAmpC in E. coli (87%), blaSHV in Klebsiella (84.8%) and Enterobacter (48.2%), blaOXA in Pseudomonas (66.6%) species. Majority of the isolates with confirmed ESBL phenotype carried blaCTX-M G1 gene (72.5%). The blaACC and blaMOX genes were not detected in the canine microbiota. The efficacy of antibiotics against bacterial infections is decreasing with rise in antimicrobial resistance, thus, there is a need to search for novel probiotic strains as potential alternatives to antibiotics. In India, there are no probiotics available for canine usage, as they are host specific. In view of this, rectal swabs (67) from healthy pups were analyzed and a total of 49 (73.1%) Lactobacillus isolates were identified based on morphological, biochemical characteristics and genus specific PCR. Of these 49 isolates, 23 were found to be positive for Group IV; six for Group I, four for Group II and 16 were found to be negative for Lactobacillus group specific PCR. In vitro antibacterial activity of canine Lactobacillus isolates on test pathogens like E. coli, Klebsiella and Enterobacter species have been studied using agar well diffusion assay. Out of 49 Lactobacillus isolates, the supernatants of 20 isolates showed inhibition against majority of the test pathogens examined. The inhibition zones were large and clear against E. coli and Klebsiella spp., but limited and hazy zones were observed against Enterobacter spp. Reduction in antibacterial activity was noticed after neutralization, proteinase K and heat treatment of supernatants, suggesting that the antibacterial activity might be partly due to organic acid production and partly due to heat labile antimicrobial proteins. Nucleotide sequence analysis of genus specific PCR products of 10 selected Lactobacillus isolates that showed consistently high antibacterial activity revealed maximum sequence homology with Lactobacillus fermentum strain RCM 14 (for six isolates), Lactobacillus agilis strain 76CL (for one isolate), Pediococcus acidilactici strain G4 (for 2 isolates) and Weissella confusa strain 3W (for one isolate). In conclusion, the present study revealed alarming β-lactamase resistance in microbiota of dogs in Andhra Pradesh. Therapeutic failures may likely to occur as resistance to commonly prescribed third generation cephalosporins was observed. Lactobacillus strains of dog faecal origin were found to have potent in vitro antimicrobial action. Furthermore, the present study highlighted need for in vivo studies in India to establish probiotic potential of dog faecal Lactobacillus species in the near future.ThesisItem Open Access MOLECULAR CHARACTERIZATION OF Pasteurella multocida ISOLATES OF BUFFALOES(SRI VENKATESWARA VETERINARY UNIVERSITY TIRUPATI - 517 502. (A.P.) INDIA, 2016-12) SUJATHA, N; Lakshmi Kavitha, K(MAJOR); Subramanyam, K.V.; Srinivasa Rao, TABSTRACT: Pasteurella multocida is a Gram negative pathogen responsible for economically important diseases in ruminants especially in cattle and buffaloes. Young animals were more susceptible than adults and the carrier animals play an important role in spreading of Hemorrhagic septicaemia (HS). The present work was taken up for isolation and characterization of P. multocida from buffaloes.A total of 196 samples were collected from slaughtered animals, clinical suspected cases, field HS case and from field HS mortalities of buffaloes in Krishna district, Andhrapradesh. Primarily the suspected samples were streaked directly on Brain heart infusion (BHI) agar. The P. multocida suspected colonies were further subjected to P. multocida species specific PCR (PM- PCR) and 23 (11.73%) were found positive. Out of 23 PM-PCR positive samples 16 were isolated as pure cultures of P. multocida by morphological tests, different cultural tests and biochemical tests. Furthermore the isolates were confirmed by conducting PM-PCR using a primer pair KMT1T7-KMT1SP6 and all the 16 isolates gave positive amplification of 460 bp product. The total isolation percentage 8.16% was observed. The antibiogram of P. multocida isolates revealed 100% sensitivity to enrofloxacin, gentamicin, tetracycline, ampicillin, ceftriaxone and penicillin-G, 93.75% to chloramphenicol, ciprofloxacin, streptomycin and co-trimoxazole, followed by meropenem, aztreonam, sulphafurazole, nalidixic acid. The isolates found highly resistant to lincomycin, clindamycin, followed by sulphadiazine. The genotypic antibiotic resistance pattern of P. multocida resulted sul2 gene in 11 isolates, whereas 5 isolates had both sul1 and sul2 genes and none of the isolates harboured catA1, strA and strB genes. The capsular typing of P. multocida was conducted using Multiplex PCR, 14 isolates were classified as type A (87.5%) and two as type B (12.5%). The Cap A isolates were obtained from tonsils and Cap B isolates from whole blood and heat of field HS cases. Further the virulence genotyping of the isolates by PCR revealed that all Cap A isolates harboured (93.75%) hgbA, 100% hgbB, ompH, ptfA genes, whereas the Cap B isolates showed 100% prevalence of hgbA, hgbB, ompH, ptfA, pfhA and tbpA genes. The OMP profile of Cap A and Cap B isolates of P. multocida was analyzed on SDS-PAGE. The Cap A isolate yielded 6 polypeptide bands of approximate molecular weights of 19-76 kDa, whereas the Cap B isolate showed 6 polypeptide bands of 14.7- 80 kDa. Based on stain intensity, 38 kDa and 32.5 kDa were considered as major polypeptide bands in Cap A and Cap B isolates, respectively.ThesisItem Open Access MOLECULAR CHARACTERIZATION OF ONCOGENES IN PATHOGENIC MAREK’S DISEASE VIRUS(SRI VENKATESWARA VETERINARY UNIVERSITY TIRUPATI - 517 502. (A.P.) INDIA, 2015-10) PRATHIBHA, Y; SREEDEVI, B(MAJOR); VINOD KUMAR, N; SRILATHA, ChABSTRACT: The present study was taken up to characterize the oncogenes from Marek’s disease (MD) suspected outbreaks in Andhra Pradesh. A total of 27 blood samples and 84 tissues were collected from MD suspected cases from different poultry flocks. The affected birds showed presence of lymphomas in different organs like liver, spleen, proventriculus, kidney, ovaries, heart, lungs and nerve. Histopathological examination revealed pleomorphic infiltration of lymphoblast cells in different affected tissues which is a characteristic of Marek’s disease virus (MDV). From the suspected samples, the deoxyribonucleic acid (DNA) was extracted and subjected to Polymerase Chain Reaction (PCR) targeting a 132 bp repeat region unique for serotype-1 MD viruses. Twenty out of 27 blood samples and all the 84 tissues samples were positive in PCR yielding a 314 bp PCR product. This represents the presence of two copies of 132 bp tandem repeats characteristic of pathogenic serotype-1 MD viruses. Further PCR was standardized for two important oncogenes Marek’s EcoRI – Q (Meq) and Viral Interleukin-8 (vIL-8) and all the positive samples were showing specific 1081 and 887 bp PCR products respectively. Representative samples from different regions of Andhra Pradesh were selected and the PCR products were purified and subjected to nucleotide sequencing at Genomics corp – Xcelris, Ahmedabad. The nucleotide and the deduced amino acid sequences of Meq and vIL-8 genes of the present field strains were compared with reference and other MDV strains from Genbank. The maximum nucleotide homology of 99.5 to 99.6 % was observed with RB-1B (very virulent) and GA (virulent) strains for the Meq gene. The vIL-8 gene sequences were 99.87 % identical to virulent LS and LMS strains. In phylogenetic analysis, the field MDV’s from Andhra Pradesh clustered with virulent MDV strains and field MDV strains from Tamil Nadu and Karnataka. The amino acid sequence of Meq gene of field MDV strains showed mutation at positions 71, 77, 80 and 139 which were similar to virulent MDV strains 571, 573 and field MDV strains of Tamil Nadu and Karnataka. The amino acid sequences of vIL-8 gene of field strains showed mutation at positions 4 and 31 which were identical to virulent strains LMS and LS. The MDV strains from the present study were neither mild nor very virulent plus MDV’s and they can be categorized into either virulent or very virulent based on sequence analysis and other criteria. From PCR positive MD suspected birds, the buffy coat was collected and MDV was isolated in duck embryo fibroblast (DEF) monolayers. The characteristic cytopathic effect (CPE) with formation of plaques was observed in DEF after 3-4 blind passages. The presence of serotype -1 MDV in cell culture fluid was further confirmed by performing PCR. In conclusion, the present study established the incidence of virulent serotype -1 MDV strains in Andhra Pradesh in MD vaccinated poultry flocks. The current MD vaccines that are being used like Herpes virus of turkey (HVT) (serotype-3) and SB-1 (serotype -2) are not effective in the control of more virulent serotype-1 MDV strains. Hence, vaccination with more efficacious serotype-1 MDV vaccines like CVI988/Rispens may be recommended for effective control of Marek’s disease in India.ThesisItem Open Access MOLECULAR CHARACTERIZATION OF Pasteurella multocida ISOLATES OF BUFFALOES(SRI VENKATESWARA VETERINARY UNIVERSITY TIRUPATI – 517 502. (A.P) INDIA, 2016-12) SUJATHA, N; Lakshmi Kavitha, K (Major); Subramanyam, K V; Srinivasa Rao, TABSTRACT: Pasteurella multocida is a Gram negative pathogen responsible for economically important diseases in ruminants especially in cattle and buffaloes. Young animals were more susceptible than adults and the carrier animals play an important role in spreading of Hemorrhagic septicaemia (HS). The present work was taken up for isolation and characterization of P. multocida from buffaloes. A total of 196 samples were collected from slaughtered animals, clinical suspected cases, field HS case and from field HS mortalities of buffaloes in Krishna district, Andhrapradesh. Primarily the suspected samples were streaked directly on Brain heart infusion (BHI) agar. The P. multocida suspected colonies were further subjected to P. multocida species specific PCR (PM- PCR) and 23 (11.73%) were found positive. Out of 23 PM-PCR positive samples 16 were isolated as pure cultures of P. multocida by morphological tests, different cultural tests and biochemical tests. Furthermore the isolates were confirmed by conducting PM-PCR using a primer pair KMT1T7-KMT1SP6 and all the 16 isolates gave positive amplification of 460 bp product. The total isolation percentage 8.16% was observed. The antibiogram of P. multocida isolates revealed 100% sensitivity to enrofloxacin, gentamicin, tetracycline, ampicillin, ceftriaxone and penicillin-G, 93.75% to chloramphenicol, ciprofloxacin, streptomycin and co-trimoxazole, followed by meropenem, aztreonam, sulphafurazole, nalidixic acid. The isolates found highly resistant to lincomycin, clindamycin, followed by sulphadiazine. The genotypic antibiotic resistance pattern of P. multocida resulted sul2 gene in 11 isolates, whereas 5 isolates had both sul1 and sul2 genes and none of the isolates harboured catA1, strA and strB genes. The capsular typing of P. multocida was conducted using Multiplex PCR, 14 isolates were classified as type A (87.5%) and two as type B (12.5%). The Cap A isolates were obtained from tonsils and Cap B isolates from whole blood and heat of field HS cases. Further the virulence genotyping of the isolates by PCR revealed that all Cap A isolates harboured (93.75%) hgbA, 100% hgbB, ompH, ptfA genes, whereas the Cap B isolates showed 100% prevalence of hgbA, hgbB, ompH, ptfA, pfhA and tbpA genes. The OMP profile of Cap A and Cap B isolates of P. multocida was analyzed on SDS-PAGE. The Cap A isolate yielded 6 polypeptide bands of approximate molecular weights of 19-76 kDa, whereas the Cap B isolate showed 6 polypeptide bands of 14.7- 80 kDa. Based on stain intensity, 38 kDa and 32.5 kDa were considered as major polypeptide bands in Cap A and Cap B isolates, respectively.ThesisItem Open Access STUDIES ON BIOFILM FORMING BACTERIAL PATHOGENS OF BOVINE MASTITIS(SRI VENKATESWARA VETERINARY UNIVERSITY TIRUPATI – 517 502. (A.P) INDIA, 2016-12) SATISH KUMAR, DONTAM; Ramani Pushpa, R.N(Major); Subramanyam, K.V; Srinivasa Rao, TABSTRACT: Bovine mastitis is the most common infectious diseases of dairy cattle which is economically important in dairy industry all over the world. The main etiological agents causing bovine mastitis are Staphylococcus aureus, Streptococcus agalactiae and Escherichia coli. Antimicrobial therapy is commonly implemented for mastitis prevention and control. Restriction of antimicrobial agents gaining access to the pathogens due to the impervious nature of the biofilm encased extracellular matrix, plays a pivotal role in biofilm antimicrobial resistance. Hence, the present study was undertaken to study the prevalence of biofilm forming bacteria involved in bovine mastitis and evaluation of their antibiogram profile. A total of 81 mastitis milk samples were investigated in the present study. Out of these, 67 (82.7%) samples were positive. No pathogens were isolated from 14 (17.3%) milk samples. A total of 95 organisms were recovered. Staphylococcus species (46.9%) was the predominant bacteria isolated followed by E. coli (33.3%), Streptococcus species (29.6%), Pseudomonas species (6.17%) and Listeria monocytogenes (1.23%).All 38 isolates of Staphylococcus spp. and 24 isolates of Streptococcus species were confirmed using genus specific primers. Whereas, 27 isolates of E. coli, 17 isolates of S. aureus, 12 isolates of Streptococcus uberis, 6 isolates of S. agalactiae, 4 isolates of Streptococcus dysgalactiae and one isolate of Listeria monocytogenes were confirmed using species specific oligonucleotide primers.The antibiogram profile indicated that Staphylococcus species, E. coli and Streptococcus species showed highest resistance to ampicillin, penicillin-G and ceftriaxone+ sulbactum followed by enrofloxacin, tetracycline and oxacillin and had least resistance to amoxicillin+ clavulanic acid and gentamicin. Whereas, the antibiogram of biofilm producing bacterial pathogens revealed more resistance to antibiotics used in the present study when compared to nonbiofilm producers.The MIC of Staphylococcus isolates ranged from 7.81- 0.49 μg/ml for enrofloxacin, 0.49- 7.81 μg/ml for gentamicin, 0.49- 31.25 μg/ml for amoxicillin, 0.49- 125 μg/ml penicillin-G, 3.9- 1000 μg/ml for oxacillin and 0.49- 31.25 μg/ml for ceftriaxone. The observed MIC of E. coli isolates ranged from 0.49- 31.25 μg/ml for gentamicin, 0.49- 125 μg/ml for amoxicillin, 3.9- 1000 μg/ml for penicillin-G, 0.49- 250 μg/ml for nalidixic acid and 0.49- 1.95 μg/ml for ceftriaxone. All isolates of E. coli were inhibited at a concentration of 0.49 μg/ml for enrofloxacin.The genotypic antibiotic profile of E. coli and Staphylococcus species was investigated. Among the 38 isolates of Staphylococcus species, 33 (86.8%) and 28 (73.7%) were carrying mecA and blaZ genes respectively. Both mecA and blaZ genes were found in 25 (65.8%) isolates of Staphylococcus species. Out of 17 isolates of S. aureus, mecA and blaZ genes were detected in 13 (76.5%) isolates each respectively. Both mecA and blaZ genes were found in 10 (58.8%) isolates of S. aureus. Tetracycline resistance genes were found in 74.07% of E. coli isolates in the present study. Among tetracycline resistance genes, tetB was found in higher frequency of 51.8%, and tetA was found in 48.1%. Whereas 25.9% isolates carried both tetA and tetB genes. None of the E. coli isolate was positive for quinolones gene.The biofilm formation of bacterial isolates was detected by using congo red agar method (CRA), microtitre plate assay (MTPA) and polymerase chain reaction (PCR). In E. coli, the frequency of biofilm producers was 66.7% when identified by CRA method, while it was 77.7% using MTPA method and by PCR, 88.8% of isolates were positive for biofilm gene (luxS). Out of 38 isolates of Staphylococcus species, 17 (44.7%) were producing biofilm by CRA method, 35 (92.1%) by MTPA. icaA and icaD genes responsible for biofilm formation in S. aureus were detected in three (17.6%) and seven (41.2%) of S. aureus isolates respectively. Two (11.76%) strains of S. aureus carried both icaA and icaD genes. None of the Staphylococcus isolates were positive to icaBC and bap genes. Out of 24 Streptococcus isolates, 12 (50%) were positive for biofilm production by CRA method, whereas 19 (79.2%) by MTPA method. Among 12 S. uberis isolates, biofilm formation was detected in 10 (83.3%) by CRA method, all 12 isolates were biofilm producers by MTPA method and the luxS gene responsible for biofilm production was detected in five (41.7%) isolates.The results of nucleotide sequences of biofilm genes of S. aureus (icaD), E. coli (luxS) and S. uberis (luxS) when blasted with reference strains in NCBI database revealed 98%, 100% and 100% homology respectively.