Cattle are an important reservoir of Escherichia coli O157:H7 leading to contamination of food and water, and subsequent human disease. This pathogen colonizes its hosts by producing several proteins such as Tir and EspA that are secreted by a type III secretion system. These proteins play a role in colonization of the intestine, suggesting that they might be useful targets for the development of a vaccine to reduce levels of this organism in cattle. Vaccination of cattle with proteins secreted by E. coli O157:H7 significantly reduced the numbers of bacteria shed in feces, the numbers of animals that shed, and the duration of shedding in an experimental challenge model. Vaccination of cattle also significantly (P=0.04) reduced the prevalence of E. coli O157:H7 in a clinical trial conducted in a typical feedlot setting. This strategy suggests it is possible to vaccinate cattle to decrease the level of E. coli O157:H7 shedding for the purpose of reducing the risk of human disease.

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An expression library was constructed from an Actinobacfllus pleuropneumoniae serotype 1 clinical isolate using a plasmid vector. The library was screened with serum raised against the culture supernatant of this strain. One Escherichia coli transformant which also reacted with convalescent serum was isolated and found to express a protein with an electrophoretic mobility of approximately 50,000. The A. pleuropneumoniae-* Corresponding author.

An expression library was constructed from ActinobaciUlus pleuropneumoniae serotype 7. Escherichia coli transformants expressing recombinant proteins were identified by immunoscreening with porcine convalescent serum. One transformant expressing a 60-kDa protein (60K protein) in aggregated form was identified. Serum raised against the recombinant protein recognized a polypeptide with an indistinguishable electrophoretic mobility in theA. pleuopneumoniae wild type after iron-restricted growth only. The recombinant protein bound transferrin after blotting onto nitrocellulose. Using a competitive enzyme-linked immunosorbent assay (ELISA), the specificity of this binding for the amino-terminal half of iron-saturated porcine transferrin was established. Also, the 60K wild-type protein bound hemin as assessed by hemin-agarose chromatography. Hemin could inhibit transferrin binding of the recombinant protein in the competitive ELISA, whereas hemoglobin and synthetic iron chelators failed to do so. Southern blot analysis of several other A. pkuropneumoniae strains indicated that highly homologous sequence is present in eight of eight isolates of serotype 7 and in some isolates of serotypes 2, 3, and 4.

The gene encoding the ActinobaciUus pleuropneumoniae serotype 1 transferrin-binding protein (tfbA) was cloned, and the carboxy-terminal 70%o of the protein was expressed as an aggregate protein in Escherichia coli.The nucleotide sequences of the aJbA genes from A. pleuropneumoniae serotypes 7 (G.-F. Gerlach, C. Anderson, A. A. Potter, S. Klashinsky, and P. J. Willson, Infect. Immun. 60:892-898, 1992) and 1 were determined, and a comparison revealed that they had 65% sequence identity. The deduced amino acid sequences showed a sequence agreement of 55%, and both proteins possessed a lipoprotein-like signal sequence. The serotype 1 TfbA protein had a predicted molecular mass of 65 kDa, compared with 60 kDa for the serotype 7 TfbA protein, and both proteins were immunologically distinct as assessed in a competitive enzyme-linked immunosorbent assay. Southern hybridization and Western blot (immunoblot) analysis of the 13 A. pleuropneumoniae type strains revealed that serotypes 2, 3, 4, 8, 9, 10, and 11 encode and express a TfbA protein highly homologous to that ofA. pleuropneumoniae serotype 7 whereas the TfbA proteins and the encoding genes of serotypes 6 and 12 were highly homologous to that found in A. pleuropneumoniae serotype 1. The aJbA genes of A. pleuropneumoniae serotypes 5A and 5B were recognized, under medium-stringency hybridization conditions, by the A. pleuropneumoniae serotype 1-derived tJbA probe, and the respective proteins were weakly reactive with the antibody raised against the A. pleuropneumoniae serotype 7 TfbA protein.