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Lala Lajpat Rai University of Veterinary & Animal Sciences, Hisar

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2008 - 20093
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Subject: Veterinary Immunology × End date: 2009 × Start date: 2004 ×
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Now showing 1 - 3 of 3
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    A study on surface immunogens of biofilm-associated Staphylococcus aureus in mice
    (LUVAS, 2008) Sharma, Krishan; Ajit Singh
    Staphylococci are the most important of all the pathogens causing clinical and subclinical bovine mastitis worldwide. In past two decades, role of ‘biofilms’ in persistence of these organisms in the host has clearly been established, but how immune system sees the biofilm-forming organisms in the infected udder remains largely unanswered. The present study was therefore conducted with the objectives to determine the immunogens specifically associated with biofilm-forming S. aureus phenotype and to assess their role in protection against biofilm-forming pathogen in the mouse model of mastitis. S. aureus isolate from a clinical bovine mastitis case was grown as planktonic and biofilm producing bacteria under different conditions. Biofilm production by S. aureus isolate was confirmed by appearance of black colonies on Congo red agar plate, Gram staining, biofilm quantitation and scanning electron microscopy. Extracellular polysaccharide (EPS) and cell membrane (CM) -xxviiantigens were extracted from the biofilm phenotype and CM antigens from the planktonic. Polypeptide profiles were determined by SDS-polyacrylamide gel electrophoresis. Several up- and down-regulated proteins in biofilm-forming S. aureus CM profile were visible. Different groups of mice (n=8/group) were inoculated with these antigens using Freund’s incomplete adjuvant (FIA) and CpG motifs-containing chromosomal DNA as adjuvant. Sequential sera samples at day 0, 14, 28, 42, 56 were collected and kinetics of humoral immune response was determined by indirect ELISA. Antigen profiles of CM were determined by immunoblotting using sera collected above. Mice immunized twice four-weeks apart were challenged with 106 CFUs of biofilm-forming S. aureus by intra-mammary route after >8 weeks in all groups for determining protection levels. Antibody (Ab) levels in biofilm-associated CM-CpG group were higher at most days than those in CM-FIA group. IgM titre, but not IgG rose in EPS groups. Planktonic CM-CpG induced lower Ab levels than CM-FIA did. Biofilm-associated CM immunogens induced lower Ab levels than planktonic CM, irrespective of adjuvant used. However, these differences did not correlate with protection against challenge. In immunoblotting, seven major antigens specific to biofilm CM were revealed. Mice immunized with biofilm-associated CM with both adjuvants withstood challenge showing 100% protection, whereas planktonic CM immunized group with both adjuvants also withstood challenge showing nearly 100% protection. ELISPOT assay detected the presence of B lymphocyte clones secreting specific antibodies against various antigens used in the study. Based on these findings, use of biofilm-associated S. aureus surface immunogens as vaccine for control of mastitis was envisaged.
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    A study on antigenic profiles of vaccine strain and field isolates of pasteurella multocida B:2
    (LUVAS, 2008) Saini, Deepak; Kadian, S.K.
    Haemorrhagic septicaemia (H.S.) is an acute infectious disease of domestic animals, particularly bovines caused by Pasteurella multocida serotypes B:2 and E:2. P. multocida is a gram-negative, coccobacillus, bipolar staining, capsulated organism which is diverse and complex with respect to antigenic variation, host predilection and pathogenesis. To date only a few potential antigens of P. multocida have been clearly defined. The various tests developed for investigation of the H.S. are still provisional and open to improvement. Extensive and in depth studies are still required for the development of a diagnostic test which can differentiate vaccinated animals from diseased ones serologically. Hence the present study was undertaken with an objective to determine total polypeptide profiles of vaccine strain and some field isolates of P. multocida B:2 and to identify the immunoreactive antigens in these profiles by immunoblotting employing antisera of vaccinated animals. Five field isolates of P. multocida B:2 and vaccine strain (P52) were grown on blood agar and then brain heart infusion broth. Whole cell lysates were prepared and total polypeptides in different isolates and vaccine strain were resolved by SDS-PAGE in 12.5% resolving gel. The polypeptide profiles so obtained were electroblotted onto nitrocellulose membrane and the blots were developed with antisera from H.S. vaccinated cattle and buffaloes. On SDS-PAGE analyses of the whole cell lysates polypeptides of different molecular weights ranging from 6l to 4 kDa were obtained. Thirteen polypeptides were common to all the field isolates and vaccine strain with little variation. Out of the 13 common bands 7 were major (61, 53, 36, 25, 21, 15 and 7 kDa) and 6 were minor (48, 41.6, 30.0, 13.2, 5.6 and 4.8 kDa). Polypeptides of molecular weight 53 and 21 kDa were minor, 5.6 kDa was major and 41.6 kDa was absent in vaccine strain (P52). On immunoblotting with sera from vaccinated buffalo immune response was observed with all sera against polypeptides of 36 and 21 kDa (Major polypeptides) and 48, 30, 13.2 and 11.5 kDa (Minor polypeptides). With vaccine strain all sera gave immune response against 60 kDa polypeptide while variable response was observed against 21, 25 and 55.5 kDa polypeptides. Immune response against polypeptides of 36 and 25 kDa (Major polypeptides) and 61.3, 13.2 and 11.5 kDa (minor polypeptides) was observed on immunoblotting with sera from vaccinated cattle. With vaccine strain all sera gave response against 36, 30 and 25 kDa polypeptide while variable immune response was observed against 21, 25 and 68.7 kDa polypeptides on immunoblotting with cattle sera. Therefore, polypeptide of 36 kDa (major polypeptide) was found to be most strongly reacting with all antisera from H.S. vaccinated animals in all isolates. This study indicates that we can not rely upon a single polypeptide antigen to be used in diagnostic test for differentiating between the diseased and vaccinated animal serologically. Moreover, a number of antigens P. multocida B:2 should be considered for inclusion in safer and efficacious vaccine formulation(s) constructed by rDNA- based methods and a subunit vaccine using any one polypeptide antigen still requires extensive and in depth studies in future.
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    Production of Staphylococcus aureus exoproteins-specific camel single domain antibodies from phage display library
    (LUVAS, 2009) Jangra, Pooja; Ajit Singh
    Phage display technology is one of the modern technologies for production of antibodies for their potential applications in diagnosis, prevention and treatment of human and animal diseases. Aim of the present study was to isolate and characterize Staphylococcus aureus exoproteins-specific dAb clones from a phage display library of LPS-immunized Indian desert camel. Total exoproteins were harvested from S. aureus culture supernatant, polypeptide profile obtained by SDS-PAGE, and SAE- p39, SAE-p27 and SAEp23 extracted from the gel. Several major bands were visible in the polypeptide profile. S. aureus exoprotein Ags-specific dAb clones were selected from the phage display library of LPS-immunized Indian desert camel by three rounds of panning. The Ag-specific dAb-displaying phages were enrichment at the end of the three rounds as revealed by phage titration, Phage-ELISA and VHH-PCR. The expression of soluble dAb.HA clones by infecting amber non-suppressor WK6 with Ag-specific phages was confirmed by SDS-PAGE and blotting. The four high-expressing VHH clones were subcloned into pHEN6c vector and the resulting 14 subclones expressed as soluble dAb.6xHis as analyzed by SDSPAGE. Only four of the 14 clones were found positive by indirect ELISA. Three of these Ag-specific clones, viz., dAb.6xHis Cl-7-1, Cl-7-5 and Cl-9-2 were expressed in WK6 and purified by Ni-chelate chromatography using Ni-TED columns or Ni-NTA resin followed by ultrafiltration through 30 kDa membrane. The three purified dAb.6xHis clones bound to total exoproteins and SAE-p39 Ags, but not SAE-p27 and SAE-p23 as detected by both indirect ELISA and immunoblotting. S. aureus β-hemolysin in the culture supernatant was neutralized completely by dAb.6xHis Cl-7-5, partially by dAb.6xHis Cl-7-1 and not at all by dAb.6xHis Cl-9-2. The dAb.6xHis Cl-7-5 resisted thermal inactivation at all six temperatures from 50°C to 99°C for neutralizing β- hemolysin. These findings have revealed that dAb Cl-7-5 has the potential for diagnostic and therapeutic applications in future.