Thesis

Browse

Search Results

Now showing 1 - 9 of 25

Open Access
BUBALINE NEUTROPHILS FUNCTIONAL ACTIVITY AND LYMPHOCYTES BLASTOGENESIS RESPONSES IN Escherichia coli INFECTION, in vitro: EFFECT OF IMMUNOMODULATION
(SRI VENKATESWARA VETERINARY UNIVERSITY TIRUPATI - 517502. (A.P.) INDIA, 2019-11) VIJAYA KAMESWARI, VEDULA; ANAND KUMAR, P (MAJOR); RAMANI PUSHPA, R.N.; BINDU KIRANMAYI, CH.
The present study was conducted to investigate the immunomodulatory activity of phytochemicals thymol and quercetin, on innate and adaptive immune responses of buffaloes in correlation to in vitro phagocytic activity of neutrophils and lymphocytes blastogenesis responses against Gram negative bacteria Escherichia coli. Neutrophils eliminate pathogens by phagocytosis which is an important host defense mechanism. An important activity of phagocytes is their ability to respond to stimuli by activation of the respiratory burst. Therefore, the phagocytic activity of neutrophils is estimated by nitro blue tetrazolium (NBT) assay. Also, the effect of phytochemicals quercetin and thymol on phagocytic activity of bubaline neutrophils during in vitro E. coli infection is studied. The phagocytic activity of neutrophils treated with different concentrations of quercetin (25 μM and 50 μM) and thymol (15 μg/ ml and 30 μg/ ml) separately, and incubated with live E. coli/ opsonized E. coli/ zymosan is found to be increased compared to their normal counterparts (without immunomodulators) with significant difference (p<0.05). The immunomodulators quercetin and thymol increased the activity of superoxide anion in neutrophils, which is reflected interms of increased absorbance that is an indicator of increased phagocytic activity. Adaptive immunity is very important as it is specific immune response directed against a targeted antigen. Among the peripheral blood mononuclear cells (PBMC), lymphocytes account for major population. Since, the proliferation is a fundamental characteristic of the lymphocytes, induction of lymphocyte proliferative responses by antigen/ mitogen in vitro with PBMC culture is referred as a representative index for cellular immunocompetence. Therefore in the present study lymphocyte transformation test or lymphocyte proliferation assay is performed by stimulating the PBMC’s of buffaloes with mitogen Concanavalin A (Con A) and antigen E. coli (heat inactivated), separately, using a tetrazolium salt XTT (2, 3-Bis-(2-methoxy-4-nitro-5-sulfophenyl]- 2H-tetrazolium-5-carboxyanilide salt) assay. In this study stimulation of immunomodulators treated PBMC with heat inactivated E. coli resulted in more stimulation index (S.I) compared to stimulation of immunomodulators treated PBMC with Con A. Antigenic stimulation appeared to stimulate the PBMC with more intensity than that of Con A stimulation. These results provided leads about the immunostimulatory role of the phytochemicals in increasing the phagocytic activity of neutrophils and proliferative responses of PBMC in in vitro cultures. Further research is required to establish the mechanism of action underlying it.
Open Access
BUBALINE NEUTROPHILS FUNCTIONAL ACTIVITY AND LYMPHOCYTES BLASTOGENESIS RESPONSES IN Staphylococcus aureus INFECTION, in vitro: EFFECT OF IMMUNOMODULATION
(SRI VENKATESWARA VETERINARY UNIVERSITY TIRUPATI - 517502. (A.P.) INDIA, 2019-11) ASHOK KUMAR, K; ANAND KUMAR, P (MAJOR); LAKSHMI KAVITHA, K; ASWANI KUMAR, K
Buffaloes are important dairy animals of Andhra Pradesh (64.33 lakhs population) that contribute 69% of the total milk produced in the state. Staphylococcus aureus is one of the the most important Gram-positive bacterial pathogen that causes pneumonia, mastitis, phlebitis, meningitis, urinary tract infections, osteomyelitis, endocarditis and superficial skin lesions such as furunculosis etc in animals. Immune response against invading pathogens is crucial for keeping up the host’s health status. The desired optimal immune response in a host may be achieved through immunomodulation. Many synthetic, microbial and natural products are reported to act as immunomodulators. Phytochemicals are naturally occurring plant derived compounds with bioactive potentials and are used as immunomodulators in the treatment and management of infections. Phagocytic activity of neutrophils and lymphocytes proliferation assay or lymphocyte transformation test with PBMC which are considered as indicators of innate and adaptive immune responses, respectively, are included in this study to investigate the immunomodulatory activity of phytochemicals curcumin and quercetin. In this study blood samples were collected from apparently clinically healthy she buffaloes (4 to 5 years of age with similar physiological status) and infected in vitro with S. aureus isolated from clinical mastitic milk sample. Phagocytic activity of the neutrophils was measured by semi quantitative nitroblue tetrazolium assay (NBT). The reduction of NBT to formazan by the superoxide anion generated in the respiratory burst is measured at 540 nm. Bubaline neutrophils are treated with different concentrations of curcumin and quercetin, separately and incubated with live S. aureus / opsonized S. aureus / zymosan. The phagocytic activity of bubaline neutrophils treated with 25 μM curcumin and incubated with opsonized S. aureus is significantly different (p<0.05) with phagocytic activity of neutrophils treated with 50 μM curcumin and incubated with opsonized S. aureus. The phagocytic activity of bubaline neutrophils treated with 50 μM curcumin and incubated with opsonized S. aureus is significantly different (p<0.05) with phagocytic activity of neutrophils treated with 50 μM of quercetin and incubated with opsonized S. aureus. The in vitro lymphocyte proliferation assay is routinely used as a measure to assess the functional status of lymphocytes. Though initially radioactive isotopes were used in cell proliferation assays due to radiation hazards associated with radio isotope assays the tetrazolium salt-based assays are employed for the proliferation assay. In the present study XTT {sodium3'-[1-[(phenylamino)-carbonyl]-3, 4-tetrazolium]-bis (4-methoxy-6- nitro) benzene-sulfonic acid hydrate} assay was used in cell proliferation assays. The stimulation index (S.I) of PBMC stimulated with Con A is significantly different (p<0.05) with the S.I of PBMC treated with 25 μM & 50 μM of quercetin and stimulated with Con A. The S.I of PBMC stimulated with Con A is significantly different (p<0.05) with the S.I of PBMC treated with 25 μM & 50 μM of quercetin and stimulated with heat inactivated S. aureus. Similarly, the S.I of PBMC treated with different concentrations of immunomodulators is significantly different (p<0.05) with other related groups. Therefore it is concluded that the phytochemicals curcumin and quercetin exhibited immunomodulatory activity on innate and adaptive immune response of buffaloes. Further research is required to establish the mechanisms of action underlying it.
Open Access
GENETIC DIVERSITY OF BOVINE, OVINE AND PORCINE ISOLATES OF Pasteurella multocida
(SRI VENKATESWARA VETERINARY UNIVERSITY TIRUPATI - 517502. (A.P.) INDIA, 2019-04) JAGADESH, J; Lakshmi Kavitha, K(MAJOR); Ramani Pushpa, R.N.; Srinivasa Rao, T
The Gram-negative bacterium, Pasteurella multocida, is an important worldwide primary and opportunistic pathogen as well as the common commensal of many wild and domesticated animals. The present work was taken up to characterize and observe genetic diversity of bovine, ovine and porcine isolates of P. multocida. A total of 283 samples were collected from apparently healthy slaughtered animals (276) and clinical cases (7). Primarily the suspected samples were streaked directly on Brain heart infusion (BHI) agar. Further when all the suspected samples subjected to PMPCR, 86 (30.3%) were found positive. Out of 86 PM-PCR positive samples, 31 were identified (bovine 8, ovine 11 and porcine 12) as pure cultures of P. multocida by morphological tests, different cultural and biochemical tests. Further the 31 isolates were finally confirmed by employing PM-PCR using a primer set KMT1T6-KMT1SP6 with 460 bp amplicon and all the isolates were found positive in PM-PCR. The total isolation percentage of 10.9% was observed. The capsular typing was done by Uniplex PCR targeting Cap A and Cap D capsule specific gene primers, yielded 20 Cap A isolates, 8 Cap D isolates and 3 untypable isolates. The virulence gene profile of isolates showed high prevalence of hgbA gene (77.41%) among the three iron binding proteins, followed by hgbB gene in 41.9% of isolates, among these lowest prevalence was observed in bovine isolates. Gene tbpA has lowest prevalence (19.35%) among the three iron binding proteins. Moreover, the tbpA gene was highly associated with ovine isolates. The higher prevalence of adhesion related gene such as ptfA (76.66%) was observed. This gene was detected 100% in porcine isolates. Dermonecrotxin gene was noticed in high percentage in ovine isolates (27.27%), while only one isolate of bovine harbored this gene. The pfhA gene revealed clear association to Cap A strains (50%), and none of the other capsule types of P. multocida harbored this gene. The dermonecrotoxin toxA was found in 25% of Cap D strains, followed by 10% of Cap A. Moreover, P. multocida strains of capsule type A, and D showed high prevalence of the ompH, ptfA and hgbA compared to cap-negative strains. Molecular typing techniques (REP-PCR, ERIC-PCR and (GTG)5-PCR) differentiated all 49 strains (isolates from this study and previous isolates) into different profiles. All the isolates were found genetically distinct from standard P. multocida strain P52 (Vaccine strain of India). Genetic diversity among the isolates of P. multocida and their genetic dissimilarity with P52 vaccine strain showed an indication about vaccine strategy. This study provided a clear evidence of presence of more than one isolate type in host species and also provided indication of high genetic variation among field isolates of P. multocida and may be the reason of vaccine failure and outbreaks. Furthermore the isolation of different capsular types from single host species warrens the need for the use of polyvalent vaccine against Pasteurella infections in their respective hosts.
Open Access
MOLECULAR CHARACTERIZATION AND PHYLOGENETIC ANALYSIS OF CANINE DISTEMPER VIRUS IN DOMESTIC DOGS
(SRI VENKATESWARA VETERINARY UNIVERSITY TIRUPATI - 517502. (A.P.) INDIA, 2019-03) KEERTHI, BANDARU; RAMANI PUSHPA, R.N.(MAJOR); LAKSHMI KAVITHA, K; SRINIVASA RAO, T
Canine distemper is a highly contagious viral disease caused by Canine distemper virus (CDV) affecting dogs and other wild carnivores throughout the world. CDV is a morbillivirus under Paramyxoviridae family causing highly systemic infection with prominent respiratory, gastrointestinal and nervous signs in dogs and other wild carnivores. The objective of the present work is to isolate and characterize CDV in domestic dogs and its Phylogenetic analysis. The study was aimed to detect CDV from clinical samples of the suspected dogs by diagnostic RT-PCR. The positive samples were subjected to virus isolation in MDCK cells and the presence of viral RNA was confirmed by RT-PCR. All the clinical samples including brain tissue were confirmed for the presence of viral RNA with the partial L gene with an expected amplicon size of 268 bp and the positive L gene cDNA samples were further amplified for H and F genes. All the three genes were sequenced and phylogenetically analyzed with the CDV strains available in GenBank. Different clinical samples viz. ocular, nasal, and fecal swabs, blood, urine and brain tissue were collected from 48 CD suspected dogs and then screened by RT-PCR. Out of 48 suspected samples, 34 (70%) were found positive. The most common clinical signs recorded in the positive cases were pyrexia (102.8-105°F), mucopurulent ocular and nasal discharges, hyperkeratosis of digital pads, vesiculo-pustular dermatitis and nervous disorders. MDCK cell line was successfully used to isolate CDV. Three number of RT-PCR positive ocular samples were propagated in MDCK cells for CDV isolation and all the three initially did not produce characteristic CPE but from third passage onwards characteristic CPE like rounding, aggregation, ballooning and clumping of chromatin material was observed. The presence of virus in all the cell culture harvests was confirmed by RT-PCR using L gene based diagnostic primers. The cell culture harvests were ultracentrifuged and the virus pellet showed amplification of L and H genes. Purification of virus was done by discontinuous sucrose density gradient centrifugation and the purified virus was subjected to SDS-PAGE for protein profiling. The bands of sizes approximately 180KDa (L), 76KDa (H), 66KDa (P), 58KDa (N) were visualized. Brain and spleen tissues were processed for histopathological examination. Lymphoid depletion and focal areas of necrosis in the white pulp of spleen was observed. Histopathology section of brain revealed infiltration of neutrophills, lymphocytes, perivascular cuffing and necrosis of neurons. The partial L, H and F gene sequencing was performed to compare the genetic variation among the field isolates and vaccine strain. All the field isolates of CDV partial L, H and F genes showed 94.6-98% of nucleotide sequence identity with that of the other CDV reference strains available in GenBank. The Onderstepoort vaccine showed 95%, 92% and 90% homology with the partial nucleotide sequences of L, H and F genes of CDV field isolates respectively. The nucleotide sequences identity of L gene among the field isolates in the present study was 98 to 99%. The CDV partial L gene field isolates showed highest similarity with the strains of Ludhiana and Japan. Whereas the partial H and F gene field isolates showed highest similarity with the strains of UP and France isolates respectively. All the field isolates showed lowest similarity with commercially available vaccine strains. The obtained sequences were also compared with the reference CDV sequences available in GenBank to establish the phylogenetic lineage. The results generated with the obtained sequences of the field isolates for L, H and F genes made a distinct clade in phylogenetic tree which was clearly separated from the commercial CDV vaccine strains and grouped with the Ludhiana, Japan, UP and France isolates. The phylogenetic tree based on partial L gene sequence revealed that the two local isolates were grouped together with the Ludhiana strain and another isolate with the Japan strain
Open Access
MOLECULAR CHARACTERIZATION OF Pasteurella multocida AND Mannheimia haemolytica FROM SHEEP PNEUMONIA
(SRI VENKATESWARA VETERINARY UNIVERSITY TIRUPATI - 517502. (A.P.) INDIA, 2019-01) VIDYA SUSMITHA, K; LAKSHMI KAVITHA, K(MAJOR); SRIVANI, M; RAMADEVI, V
Pasteurella multocida and Mannheimia haemolytica are responsible for major economic problems in ruminant production worldwide. These organisms cause respiratory diseases and shipping fever under stress in sheep. The objective of the present study is molecular characterization of Pasteurella multocida and Mannheimia haemolytica from sheep pneumonia. A total of 195 lung samples were collected and were streaked on brain heart infusion agar. The suspected colonies were subjected to P. multocida species specific PCR (PM- PCR) and M. haemolytica species specific PCR using PHSSA and Rpt2 genes by multiplex PCR and found 12 (6.15%) and 15 (7.69%) positives, respectively. Out of 12 PM-PCR positive samples, 7 pure cultures of P. multocida were obtained. Further the isolates were confirmed by conducting PM-PCR and all gave positive amplification of 460 bp product. The total isolation percentage 3.59% was observed. Among the 15 PHSSA and Rpt2 positive samples of M. haemolytica 10 were isolated as pure cultures. Later the cultures were confirmed by PCR. The isolation percentage of M. haemolytica observed was 5.12%. P. multocida capsule biosynthesis gene showed Cap A and Cap D with 28.57% and 57.14%, respectively. One isolate was untypable. The distribution of virulence genes showed pfhA (14.28%), tbpA (71.41%), ompH (57.14%), ptfA (0%), hgbA (57.14%), hgbB (0%) and toxA (57.14%). It was observed Cap D association with toxA gene and Cap A with pfhA gene. The virulence genotyping of M. haemolytica was conducted using lktA gene in which 30% of the isolates possesed this gene. The isolates of P. multocida revealed antibiotic sensitivity to gentamicin, cotrimoxazole, ceftriaxone, ampicillin, sulfamethoxazole, streptomycin, amoxicillin-clavulanic acid, nalidixic acid (100%), followed by chloramphenicol and colistin (71.42%), amoxicillin (57.1%), enrofloxacin (4.85%), clindamycin (28.57%). However the isolates were resistant or intermediately resistant to tetracycline (100%). The distribution of antibiotic resistance genes were catA1 (100%), strA (57.14%), strB (71.42%) and tetB (42.85%). The results of sensitive antibiogram pattern of M. haemolytica revealed gentamicin, cotrimoxazole, ceftriaxone (100%), ampicillin, sulfamethoxazole (90%), chloramphenicol, nalidixic acid (80%), amoxicillin-clavulanic acid (60%), tetracycline (40%), amoxicillin (3%), colistin, enrofloxacin (1%). The distribution of antibiotic resistance genes showed tetB (40%) and absence of other genes studied. The gross pathology of the infected lungs showed cranioventral consolidation and pleural adhesions. The histopathological changes observed in the affected tissues were infiltration of neutrophils and mononuclear cells into the bronchial lumen, alveoli and bronchopneumonia was evident. The research work paved way for development of conventional vaccines using different capsular types. It also paved way for future development of rapid diagnostic kits and probable recombinant vaccines using the properties of virulence genes.
Open Access
STUDIES ON ANTIMICROBIAL RESISTANCE OF Escherichia coli ISOLATES FROM LAMB DIARRHOEA CASES
(SRI VENKATESWARA VETERINARY UNIVERSITY TIRUPATI - 517502. (A.P.) INDIA, 2018-12) SUJATHA, T; Srivani, M(MAJOR); Subramanyam, K.V.; Srinivasa Rao, T
A study was carried out on the isolation, molecular characterization, and antimicrobial resistance of E. coli isolates from 1-7, 8-30, 31-60 and 61-90 day-old diarrhoeic lambs from Vizianagaram, West Godavari and Krishna districts of Andhra Pradesh. A total of 212 samples were collected, from which 170(80.18%) E. coli were isolated. Highest prevalence of E. coli was observed in West Godavari (86.41%) and Vizianagaram (82%) districts, while lowest prevalence was found in Krishna district (72.83%). Among different age groups, highest prevalence of E. coli was observed in 1-7 day-old diarrhoeic lambs (84%) while lowest prevalence was detected in 61-90 day-old diarrhoeic lambs (72.72%). Among the E. coli isolates, 87.05% were shiga toxigenic (STEC) and none of the isolates belonged to enterotoxigenic (ETEC). Among the virulence genes of STEC, eaeA &hlyA genes were highest (35.13%) followed by 12.83, 11.48, 10.13, 8.10, 5.4, 4.72 and 2.70% isolates carried stx1; hlyA; stx2;stx2&eaeA; stx1&eaeA; stx2&hlyAand stx1&hlyA and all STEC gens (stx1, stx2, eaeA&hlyA),respectively. Out of 96 hlyA carrying E. coli isolates, only seven isolates did not show any haemolysis on sheep blood agar. Highest antibiotic resistance was observed for the E.coli isolates against colistin (98.82%) and sulphamethizole (89.41%) while enrofloxacin (5.88%), gentamicin (5.33%), and chloramphenicol (1.17%) were effective. Among the STEC isolates, highest antimicrobial resistance (100%) was observed to colistin followed by sulphamethizole (95.94%), while chloramphenicol (1.35%) was effective. An ESBL phenotype was confirmed in a total of 72 STEC isolates. β lactamase genes were detected in 91.21% of STEC isolates with blaTEM being the predominant gene detected (91.21%) followed by blaCTX-M group 1 (77.70%,), blaCTX-M group 2 (10.13%) blaOXA (4.72%,), blaSHV, blaTEM+OXA, and blaCTX-9 (3.37%,), CTX-1+CTX-9 (2.02%) and SHV+OXA (1.35%), respectively. Clove oil was able to inhibit 70% and 50% of multidrug resistant E. coli by well and disc diffusion methods while cinnamic acid did not show any antibacterial activity by both the methods. The present study provides an insight on prevalence of multidrug resistant E. coli against which herbal extracts like clove oil may be effective in treating lamb diarrhoea cases.
Open Access
STUDIES ON BIOFILM INHIBITION AND ANTIMICROBIAL RESISTANCE IN S. aureus AND STREPTOCOCCUS SPECIES CAUSING BOVINE MASTITIS
(SRI VENKATESWARA VETERINARY UNIVERSITY TIRUPATI - 517502. (A.P.) INDIA, 2018-12) JOSEPH, GREESHMA ANN; RAMANI PUSHPA, R.N.(MAJOR); LAKSHMI KAVITHA, K; SRINIVASA RAO, T
Bovine mastitis is recognised as the most economically important disease affecting Dairy industry in India and all over the world. Most prevalent bacterial etiological agents identified in bovine mastitis are Staphylococcus aureus, Streptococcus species, E. coli and other Gram-negative organisms. The preferred treatment regime during the past decades for mastitis is antibiotic therapy. The associated antimicrobial resistance and recurrent infections caused by slime producing bacteria is the major constraint. The time has elapsed to think about new treatment methods and agents. Hence the present study is on the biofilm forming S. aureus and Streptococcus species causing bovine mastitis and the effect of antibiofilm agents on the antimicrobial resistance of the microorganisms. A total of 91 Bovine mastitic milk samples were examined and out of this 75 (82.41 %) samples were positive for S. aureus and Streptococcus species. A total of 110 isolates were obtained, the prevalence observed was S. aureus 62 (56.36%), other Staphylococci 2 (1.81%), S. uberis 44 (40%) and other Streptococci 2 (1.81%). All S. aureus and Streptococcus species isolates were confirmed by genus specific PCR followed by species specific PCR. Two Streptococcus isolates did not react to any of the specific primers used for S. uberis, S. agalactiae and S. dysgalactiae. Biofilm formation was detected using qualitative Congo red agar method (CRA), quantitative microtiter plate (MTP) assay and biofilm gene was detected using PCR. On CRA method 88.71% S. aureus isolates and on MTP assay 98.39 % isolates were biofilm producers. The ica locus was detected in 93.54% of the S. aureus isolates. The icaA gene was detected in 87.09% isolates and icaD was detected in 40 isolates. Fifty eight percent were carrying both icaA and icaD genes. On CRA 80.43% Streptococcus species isolates and on MTP assay 84.78% were biofilm producers. Among S. uberis isolates 34.09% were positive for the biofilm gene luxS by PCR. Twenty four isolates without luxS gene also produced very weak or moderate biofilm. On antibiotic sensitivity test the S. aureus isolates were resistant to Penicillin G, Ampicillin, Erythromycin, Methicillin, Clindamycin, Amoxycillin/Clavulanic acid, Streptomycin and were least resistant to Gentamicin, Cotrimoxazole, Chloramphenicol, Tetracycline and Vancomycin. All MRSA isolates were found to be biofilm producers while 96.87 % of MSSA were biofilm producers. MRSA was showing 2 to 4 times more resistance to all tested antibiotics than MSSA. The Streptococcus species showed high resistance to Ceftriaxone followed by Streptomycin, Erythromycin, Penicillin G, Tetracycline, Enrofloxacin, Amoxycillin/Clavulanic acid and least resistance to Gentamicin, Chloramphenicol and Ampicillin/ Sulbactam. Biofilm forming isolates were highly resistant to Ceftriaxone (66.66%), Streptomycin (41.02%), Erythromycin (38.46%), Tetracycline (33.33%) and Penicillin G (30.76%), whereas non biofilm formers showed considerably low resistance to Ceftriaxone (28.57%), Penicillin G (14.28%), Streptomycin (14.28%) and were 100% sensitive to all other antibiotics used. Biofilm inhibition studies were conducted in biofilm producing S. aureus and Streptococcus species identified by MTP. The mean±SE values of inhibition rates by 30 μg /ml Ursolic acid (UA), 100 μg /ml UA, 30 μg /ml resveratrol and 100 μg/ml resveratrol on 26 S. aureus isolates were found to be 40.67±4.64%, 62.03±3.61%, 37.38±4.86% and 53.66±4.25% respectively. Inhibition rates of antibiofilm agents on MSSA were found to be higher than MRSA except for the isolates treated with resveratrol 100 μg /ml concentration. Biofilm inhibition studies were conducted in 31 Streptococcus isolates. The mean±SE values of inhibition rates by 30 μg /ml UA, 100 μg /ml UA, 30 μg /ml resveratrol, 100 μg /ml resveratrol was 33.96±3.17%, 57.40±2.8%, 31.35±3.12% and 46.28±3.47%, respectively. Also, antimicrobial resistance of the isolates treated with antibiofilm agents at concentrations of 100 μg /ml UA and 100 μg /ml resveratrol for 18h was found to be decreased by at least 50% for each antibiotic.
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, A
ABSTRACT: 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 blue
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, K
ABSTRACT: 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.

Thesis

Browse

Thesis Type

Type

Author

Date

Subject

Search Results