Purification and characterization of the Pasteurella haemolytica 35 kilodalton periplasmic iron-regulated protein.

Pasteurella haemolytica serovar A1 is the causative agent of acute fibrinohemorrhagic pneumonia also known as shipping fever. Many pathogens, including P. haemolytica, survive in their respective hosts through the up-regulation of an iron acquisition system. In this study we identified, purified and characterized a 35-kDa periplasmic iron-regulated protein. The N-terminal sequence of the iron-regulated protein ANEVNVYSYRQP YLIEPMLK was identical to the deduced amino acid sequence of the ferric binding protein, FbpA, of P. haemolytica. Growth of P. haemolytica in a synthetic medium (RPMI-1640), without iron and supplemented with 50 gM 2,2' dipyridyl, facilitated the expression, isolation and purification of the native P. haemolytica FbpA. The protein was purified to homogeneity by using ammonium sulfate precipitation followed by CM-Sepharose ion exchange chromatography. SDS-PAGE showed a single band with a molecular weight of 35,369. Isoelectric focusing showed multiple bands with pIs of 5.5, 5.6, 5.8, and one major band with pI of 6.4. The molecular weight obtained by electrospray mass spectrometry was 35,822. Equilibrium velocity ultracentrifugation established that the protein existed as a monomer under native conditions with an apparent molecular weight of 33,795. Analysis of secondary structure of FbpA by circular dichroism showed 42.1% alpha helical structure. This protein is the second periplasmic iron-regulated protein described for P. haemolytica.

Differentiation by western blotting of immune responses of cattle vaccinated with Brucella abortus strain 19 or infected experimentally or naturally with virulent Brucella abortus.

Brucella abortus strain 19 salt-extractable proteins fractionated by differential ammonium sulfate precipitation were used in a western blotting method to detect bovine immunoglobulin G antibodies to B. abortus. Sera from infected cattle and from cattle vaccinated with strain 19 and subsequently exposed to virulent B. abortus bound to a common group of antigens ranging in molecular weights from 31,000 to 45,000 daltons. Immunoglobulin G antibodies in sera from the latter group in addition also bound to antigens with molecular weights of 66,000 to 71,000 daltons. Some sera from cattle vaccinated when sexually mature reacted similar to those from infected cattle, while immunoglobulin G antibodies in sera from Brucella-free cattle and vaccinated calves did not bind to either group of antigens. In general, fractionation of the proteins by ammonium sulfate precipitation offered no advantage for detecting differences between groups of sera. Ammonium sulfate fraction 0 to 35% reacted with a larger number of sera from a naturally infected group than fraction 0 to 70%. Both fractions reacted equally well with sera from the other groups of cattle, while fractions 35 to 70% and 70 to 100% reacted poorly in this technique. The attractive feature of the blot is that sera from calfhood-vaccinated cattle did not react.

Immunogenicity of Brucella-extracted and recombinant protein vaccines in CD-1 and BALB/c mice.

A study was conducted to determine whether subcomponent proteins (previously identified as BCSP20, BCSP31, and BCSP45, and the corresponding recombinant proteins rBCSP20, rBCSP31, and rBCSP45) that were recovered from the cell surface of Brucella abortus strain 19 were immunogenic and protective for mice when compared with Brucella cell surface protein (BCSP) and with a proteinase K-treated lipopolysaccharide (PKLPS) extracted from B abortus strain 2308. Protection was evaluated after challenge exposure with a virulent culture of B abortus strain 2308, using CD-1 or BALB/c mice or both inoculated with vaccines of various combinations and concentrations, with and without PKLPS or BCSP. Protection was assessed by enumeration of splenic colony-forming units, reduced mean splenic weight relative to controls, and the relative serologic responses (immune response) in an ELISA. The general results indicate that BCSP, PKLPS, BCSP20, and BCSP31 are immunogenic or protective or both. Protectiveness was not observed for each of the recombinant proteins; however, results from the combined recombinant protein vaccine study suggest the immunogenicity of the recombinant proteins. The apparent immune-inducing properties of BCSP20 and BCSP31 are thought to be attributable to the presence of an immunogenic and protective BCSP fraction (possibly lipopolysaccharide) still associated. Serologic results support our conclusion that each of the recombinant protein vaccines did not induce a protective response comparable to that of BCSP or PKLPS, even when the subcomponents were combined. Although the results suggest that the subcomponents of BCSP apparently induced partial protection, they are thought to be only a part of the antigens contained in BCSP that influence the serologic response.(ABSTRACT TRUNCATED AT 250 WORDS)

Effects of proteolytic enzymes on mammary tumor growth: in vitro and in vivo suppression and cellular death of tumor cells.

Proteolytic enzymes may have potential value in the prophylaxis of malignant tumor development. C3H/HEJ mice, used for their ability to produce spontaneous mammary tumors, were injected intraperitoneally (IP) with proteolytic enzyme hydrolysate at a dosage range of 0.038 to 0.462 mg/gm body weight. The injections were given every other day, once a day for six months. The pathology results showed suppression of growth, and necrosis (and in some cases encapsulation) of the mammary tumors in C3H/HEJ mice. Concurrently, SP 2/0-AG 14 cells grown in the presence of 0.25 mg enzyme/ml to 3.75 mg enzyme/ml of proteolytic enzymes, showed little cellular deterioration when the dosage range remained below 1 mg enzyme/ml. When dosage ranges were greater than 1 mg enzyme/ml, cellular necrosis occurred within three days of the addition of the proteolytic enzymes. These results demonstrate that the proteolytic enzymes used in these experiments were beneficial in preventing tumor development and prolonging survival of C3H/HEJ mice when used in the appropriate concentration range. A portion of these results were presented elsewhere (2nd Int. Biotechnol. Expo; Oct. 1989; San Francisco).