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ThesisItem Open Access PHENOTYPIC AND GENOTYPIC CHARACTERIZATION OF METHICILLIN SENSITIVE AND RESISTANT Staphylococcus aureus (MSSA & MRSA) ISOLATED FROM BOVINE MASTITIS(College of Veterinary Science, Assam Agricultural University, Khanapara, Guwahati, 2020-12) ALI, ARFAN; SAIKIA, G. K.The present study was undertaken on characterization of Staphylococcus aureus isolated from bovine mastitic milk in respect of their phenotypic and genotypic characteristics more particularly resistance to methicillin (MSSA & MRSA) and other groups of antimicrobial agents, presence of methicillin resistance and other virulence genes. To carry out the study, a total of 1328 quarter milk samples from 812 animals of organized and unorganized dairy farms of Kamrup (M), Kamrup (R) and Nalbari districts of Assam were screened by California Mastitis Test (CMT) out of which 630 animals (1328 quarter) were found positive for mastitis. The 630 mastitic animals comprised 117 clinically and 513 subclinically affected dairy cows. The overall prevalence of mastitis including clinical (14.41%) and subclinical form (63.18%) mastitis in these three districts was 77.59%. Maximum number of animals had infection involving two quarters in both clinical (47.86%) and subclinical (52.44%) mastitis. Involvement of right hind quarters was higher (28.91%) than the left hind quarters (28.13%) in clinical mastitis, while it was higher in left hind quarters (29.10%) than right hind quarters (26.21%) in subclinical mastitis. Higher prevalence rate of clinical (15.36%) and subclinical (68.76%) mastitis was recorded in organized farms in comparison to clinical (12.13%) and subclinical (49.79%) mastitis in unorganized farms. A total of 194 isolates of staphylococci were obtained from 630 bovine mastitic milk, out of which 151 (77.84%) coagulase-positive isolates identified as Staphylococcus aureus by phenotypic tests were confirmed genotypically by detection of S. aureus specific aroA gene by PCR. Of the 151 isolates, 54 (35.76%) were from clinical and 97 (64.24%) from subclinical mastitis and all of them produced coagulase and fermented mannitol. The prevalence of S. aureus associated mastitis was found to be 46.15% and 18.91% for clinical and subclinical forms, respectively. The prevalence of MRSA was 9.27% (14) as determined by cefoxitin resistance in phenotypic tests and confirmed by detection of mecA gene by PCR. The MRSA isolates were completely resistant (100%) to methicillin, cloxacillin, cefoxitin, tetracycline, streptomycin, colistin and mupirocin followed by higher degree of resistance to gentamicin and oxytetracycline (85.71% each) and moderate resistance to neomycin (50%). The MSSA isolates exhibited higher degree of sensitivity (73.72 – 100%) to tetracycline, amoxyclav, cefotaxime, ciprofloxacin, colistin, neomycin, streptomycin, mupirocin, ceftriaxone, gentamicin, cloxacillin, oxytetracycline, teicoplanin except cefepime to which they were least sensitive (54.01%). Out of 151 S. aureus isolates, 55 (36.42%) were multidrug resistant (MDR) which exhibited resistance against 4-12 antimicrobial agents. Among the MDR isolates, 14 (25.45%) were MRSA which showed resistance to 9-12 antimicrobial agents. A comparative study on antimicrobial resistance spectrum of MRSA and MSSA strains was conducted by disc diffusion and E-test using 10 antimicrobial agents which included penicillin, ampicillin, oxacillin, amoxyclav, cefoxitin, cefotaxime, ceftriaxone, gentamicin, ciprofloxacin and teicoplanin. All the MRSA isolates (14) exhibited similar pattern of resistance to all the agents except cefotaxime to which three isolates showed variation. All of the 38 representative MSSA isolates were sensitive to cefoxitin, oxacillin and teicoplanin in both the tests. One to three isolates showed variation in resistance pattern to rest of the antimicrobial agents. The E-test was found to be more effective than disc diffusion method for determining sensitivity of clinical isolates to antimicrobial drugs. In phenotypic characterization, all the coagulase positive isolates (MSSA and MRSA) caused alpha or beta haemolysis on sheep blood agar and showed susceptibility to novobiocin and resistance to polymyxin B which are typical characteristics of S. aureus. All the 151 S. aureus isolates harboured the virulence associated nuc (thermonuclease) and spa (staphylococcal protein A) genes and lukF-PV by six (6) and bap by two (2) isolates as revealed by PCR assay. The isolates which showed presence of lukF-PV and bap genes were methicillin resistant strains of S. aureus (MRSA).ThesisItem Open Access CHARACTERIZATION OF OUTER MEMBRANE VESICLES (OMVs) OF Pasteurella multocida OF AVIAN ORIGIN(College of Veterinary Science, Assam Agricultural University, Khanapara, Guwahati, 2020-01) Gogoi, Anamika; Sharma, R. K.The Fowl Cholera, an infectious disease of poultry, waterfowl and many other birds is caused by Pasteurella multocida. To overcome those hurdles in poultry industry, focus has been given to identify immunogenic subcomponent of the causative agent and their use in development of modern vaccines. The present study was undertaken with a view to evaluate immunogenic potential of Outer Membrane Vesicles (OMVs) of Pasteurella multocida as well as their release under the influence of various environmental and physico-chemical factors. The extraction of OMV fraction was made from a highly pathogenic strain of P. multocida capsular type A associated with Fowl Cholera. The release of OMVs by the selected isolates was found to be significantly (p˂0.001) highest under the influence of iron deficient condition (2, 2 bipyridyl), exhibiting a protein concentration of 18.3 mg/ml. Similarly, the influence of pH in iron restricted environment was also have an impact on OMV release, which was found to be significant (p˂0.05) in reverse direction. A positive correlation could also be made in respect to the oxidative and antibiotic stress with release of OMVs. The comparative protein profiling of OMVs, OMPs and whole cell lysate of the selected pathogenic P. multocida type A isolate could exhibit more distinct and prominent protein bands in OMV fraction. The OMV fraction could also reveal the ompA (37.7-38.1 kDa), which was not prominently observed in other two fractions. The immunogenic potential of the extracted OMV fraction revealed an increasing trend of the mean antibody titre in both the immunized groups, with (Group I) or without (Group II) booster. The immunized birds of group I exhibited a significantly rising trend (p<0.05) of the mean serum antibody titre from the day of the vaccination, until it reached its peak (5947.41±62.6). The peak titre was observed on 28th day of post primary immunization, following booster on 21st day post immunization. Similarly, the immunized birds of group II the mean serum antibody titre of 7th dpi was continued to increase significantly at every weeks of observation till it reached peak on 21st (4576.27±42.9). The declining trend of the mean serum antibody titre was observed in the birds of group II from the day 28th of post immunization (4219.12±64.5) and continued till end of the study, i.e. the 60th dpi (3813.83±148.5). No significant difference could be observed between the two preparations, with and without booster in respect to the mean serum antibody titre till 21st dpi. Challenge trial could establish 100 per cent protection of vaccinated birds against homologous challenge, while development of clinical signs in the immunized birds was observed, following heterologous challenge. There was no significant difference between OMVs administered group and control group was observed in terms of blood SOD and GPx activity.
