V.K. Gupta

Invasive Escherichia coli vaccines expressing Brucella melitensis outer membrane proteins 31 or 16 or periplasmic protein BP26 confer protection in mice challenged with B. melitensis.

Because of the serious economic and medical consequences of brucellosis, efforts are to prevent infection of domestic animals through vaccines. Many disadvantages are associated with the current Brucella melitensis Rev.1 vaccine prompting development of alternative vaccines and delivery. Escherichia coli (DH5α) was engineered to express a plasmid containing the inv gene from Yersinia pseudotuberculosis and the hly gene from Listeria monocytogenes. These recombinant invasive E. coli expressing B. melitensis outer membrane proteins (Omp31 or 16) or the periplasmic protein BP26 were evaluated for protection of mice against virulent B. melitensis. Importantly, these invasive E. coli vaccines induced significant protection against B. melitensis challenged mice. Invasive E. coli may be an ideal vaccine platform with natural adjuvant properties for application against B. melitensis since the E. coli delivery system is non-pathogenic and can deliver antigens to antigen-presenting cells promoting cellular immune responses.

Antigenic characterization of Mycobacteriumbovis BCG soluble antigens

The proteins in Mycobacterium bovis BCG sonicated antigens were separated by gel filtration chromatography. A total of 6 regions were observed. All the regions were analyzed separately for their antigenic reactivity using enzyme linked-immunosorbent assay (ELISA), delayed type hypersensitivity (DTH) skin testing and an in vitro lymphocyte transformation test (LTT). Region I of high molecular weight was found to have more reactivity in comparison to regions of lower molecular weight. When region I was subjected to anion exchange chromatography, it resolved into 5 further regions. Of these regions only region III was found to have highest reactivity when tested in ELISA, DTH and LTT. This was interpreted to indicate that region III of anion exchange chromatography has immunodominant epitopes. On sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) analysis, this region was found to be a homogeneous polypeptide of 71 kDA molecular weight. All regions were found to crossreact with various mycobacteria used in this study.

Haptoglobin and serum amyloid A as putative biomarker candidates of naturally occurring bovine respiratory disease in dairy calves

Bovine respiratory disease (BRD) is one of the leading causes of morbidity and mortality in dairy calves. Identification of reliable biomarkers of naturally occurring BRD is essential for ensuring early diagnosis and treatment of calves and monitoring treatment efficacy. This need is punctuated, especially in mild to moderate cases that would greatly help to decrease recurrence and the overall prevalence of BRD. The present study was conducted to investigate the changes in serum concentrations of haptoglobin (Hpt) and serum amyloid A (SAA) and association between oxidative stress and acute phase proteins (APPs) in BRD. Hpt and SAA levels significantly increased (P < .01) in BRD stressed calves as compared to healthy subjects. There was a significant decrease (P < .01) in serum albumin (Alb) concentration of infected calves as compared to controls. The oxidative stress markers revealed a significant (P < .01) increase in lipid peroxidation (LPO) and a concurrent decrease in activities of superoxide dismutase (SOD), reduced glutathione (R-GSH) and catalase (CAT) in BRD. A significant correlation among APPs, extent of oxidative stress and clinical score (CS) of calves was depicted. A stepwise decrease in Hpt and SAA and increase in Alb was observed in infected calves post-treatment. These results suggest implication of oxidative stress in enhancing APPs and monitoring of APPs as a potential complement to clinical assessment of treatment in calves with naturally occurring BRD. Hpt may be useful as the most sensitive biomarker in BRD. However, the combined use of Hpt and oxidative stress biomarkers would greatly improve the diagnostic accuracy.

Antigenic characterization of Mycobacterium bovis BCG culture filtrate

Mycobacterium bovis BCG culture filtrate, on gelfiltration chromatography revealed four prominent regions. Of these 4, region II possessed the highest antigenic reactivity by enzyme-linked immunosorbent assay (ELISA) and delayed type hypersensitivity (DTH) skin testing in guinea pigs. On anion-exchange chromatography, region II was resolved into 5 prominent fractions of which fraction II was found to have the highest antigenic reactivity. On sodium dodecylsulphate-polyacrylamide gel electrophoresis (SDS-PAGE), M. bovis BCG culture filtrate revealed 11 structural polypeptides. Fraction II yielded a homogenous polypeptide of 15.3 kDa molecular weight. All fractions cross-reacted with various mycobacteria used in this study except a 15.3 kDa polypeptide which was specific to M. bovis BCG.