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A genomic fragment of Pasteurella baemolytica biotype A coding for a serotype 1 -specific agglutinating antigen was used as a probe in a series of hybridization experiments to determine distribution of the fragment in various P. haemolytica serotypes as well as other bacteria. Results showed presence of the fragment in seven out of the 12 serotypes tested, all of which belonged to biotype A. Two other serotypes belonging to biotype A, all three serotypes belonging to biotype T, two Pastewella multocida isolates and Escberichia coli did not have the fragment in their genome.Thus the expression of the P. haemolytica biotype A serotype I-specific agglutinating antigen (PHAlSAA) seems to be due to serotype-specific regulation of protein expression rather than to genetic deletion. Differences in methylation of the PHAlSAA-coding fragment was also noted in DpnI and Sau3AI genomic DNA digests from the various serotypes analyzed by Southern blot.However, no apparent correlation was observed between methylation and PHAlSAA expression. E. coli with a recombinant plasmid containing a homologous genomic fragment derived from P. haemolytica serotype 2 also expressed PHAlSAA.

An ssa1-homologous genomic fragment cloned from Pasteurella haemolytica serotype 2 (ST2) enabled transformation of Escherichia coli DH5␣ to a serotype 1 (ST1) phenotype through expression of the ST1-specific antigen (Ssa1). The Ssa1 protein expressed by ssa1-transformed E. coli was susceptible to heat and protease treatment and was distinct from P. haemolytica ST1-specific capsular polysaccharide. Electrophoretic analysis of in vitro-translated proteins, as well as the predicted amino acid sequence, demonstrated that Ssa1 proteins encoded from either ST1-or ST2-derived ssa1 genes were essentially identical. A comparison of the nucleotide sequences of ssa1 genes derived from P. haemolytica ST1 and ST2 revealed greater than 99% homology. Amino acid sequence homology of the predicted products of ST1-and ST2-derived ssa1 genes was greater than 98%. Northern (RNA) blot studies revealed that the presence of an increased level of ssa1 transcript in P. haemolytica ST1 grown as surface-adherent cultures on solid medium was correlated with a serologically detectable Ssa1 protein. Expression of the ssa1 transcript in ST1 was similarly upregulated by a high iron concentration in the growth medium.

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Pasteurella haemolytica biotype A, serotype 1 (Al) has been established as the primary agent responsible for the clinical disease and pathophysiologic events leading to acute lobar fibrinonecrotizing pneumonia associated with bovine pneumonic pasteurellosis(1,2,3). The bacteria produces several potential virulent factors, of which the leukotoxin (LKT) has received the most attention(4). The LKT is a heat-labile proteinaceous exotoxin, that is oxygen-stable, non-dialyzable, water soluble, and is produced in high concentrations by P.haemolytica during the logarithmic phase of growth(5). The genes that code for the synthesis and secretion of this LKT have been cloned(6). All 15 serotypes of P. haemolytica produce LKT(7). It has a molecular weight of 101-105kDa when determined by sodium dodecyl sulfate-polyacrylamide gel electrophoretic analysis(8). Attempts to purify the LKT has been futile because it is tightly bound to the LPS and there is no efficient way to remove the LPS without inactivating the LKT. It is one of a family of RTX(repeat toxins)-pore forming cytolysin which has a unique specificity, in that it is only cytocidal to ruminant leukocytes(9). This cytotoxicity is caused by the formation of pores in the cell membrane which allows the influx of calcium and results in a sequence of cell-damaging events(lO). Since this organism produces disease only in ruminants, this would support the role of LKT as a virulence factor. Indeed, several observations point to the central role that LKT has in the pathogenesis of pneumonic pasteurellosis. For example, in experimental pasteurellosis, the clinical and pathophysiologic events are dose-dependently reproduced by the intatracheal administration of live logarithmic phase P. haemolytica and not by stationary phase organisms(ll). This enhanced pathogenicity may be related to the amount of LKT produced by these different populations. Indeed, we (5) and others(12)* have shown that the logarithmic phase cells produce far greater amounts of this LKT than stationary cells. In other studies, cattle with high LKT neutralizing antibody titers have higher survival rates in the natural disease and experimental pasteurellosis than animals with low antibody titers(13). Although advances have been made in describing the cytolytic properties of the P.haemolytica LKT, the mechanisms by which it brings about lung injury in cattle are poorly understood. Our laboratory has long been interested in elucidating the contributions and mechanisms by which the LKT induce this acute lung injury in pneumonic pasteurellosis. Three experiments were designed to study if the LKT contributed in the genesis of lung injury in pneumonic pasteurellosis.

Bovine respiratory disease (BRD) remains a major economic problem for both the beef and dairy cattle industries in the U.S. (1). Recent estimates of the losses due to BRO exceed a billion dolars annually which is greater than all other diseases combined (2). The most common sequel observed in BRD is a fulminating fibrinonecrotic pleuropneumonia called shipping fever or pneumonic pasteurellosis. Although BRD is a multifactorial disease involving interaction between a number of environmental factors, various viruses and bacteria, Pasteurella haemolytica biotype A serotype 1 (A1) also called serotype 1 (ST1) has been established as the primary agent involved in the pathogenesis of the pneumonia (3). P. haemolytica ST1 can be isolated in pure culture from typical pneumonic lesions of the disease (4).