Distribution, gene sequence and expression in vivo of the plasmid encoded fimbrial antigen of Salmonella serotype Enteritidis.

The pefA gene which encoded the serotype associated plasmid (SAP) mediated fimbrial major subunit antigen of Salmonella enterica serotype Typhimurium shared genetic identity with 128 of 706 salmonella isolates as demonstrated by dot (colony) hybridization. Seventy-seven of 113 isolates of Typhimurium and individual isolates of serotypes Bovis-morbificans, Cholerae-suis and Enteritidis phage type 9b hybridized pefA strongly, whereas 48 isolates of Enteritidis hybridized pefA weakly and one Enteritidis isolate of phage type 14b failed to hybridize. Individual isolates of 294 serotypes and 247 individual isolates of serotype Dublin did not hybridize pefA. Southern hybridization of plasmids extracted from Enteritidis demonstrated that the pefA gene probe hybridized strongly an atypical SAP of 80 kb in size harboured by one Enteritidis isolate of phage-type 9b, whereas the typical SAP of 58 kb in size harboured by 48 Enteritidis isolates hybridized weakly. One Enteritidis isolate of phage type 14b which failed to hybridize pefA in dot (colony) hybridization experiments was demonstrated to be plasmid free. A cosmid library of Enteritidis phage type 4 expressed in Escherichia coli K12 was screened by hybridization for the presence of pef sequences. Recombinant clones which were deduced to harbour the entire pef operon elaborated a PEF-like fimbrial structure at the cell surface. The PEF-like fimbrial antigen was purified from one cosmid clone and used in western blot experiments with sera from chickens infected with Enteritidis phage-type 4. Seroconversion to the fimbrial antigen was observed which indicated that the Enteritidis PEF-like fimbrial structure was expressed at some stage during infection. Nucleotide sequence analysis demonstrated that the pefA alleles of Typhimurium and Enteritidis phage-type 4 shared 76% DNA nucleotide and 82% deduced amino acid sequence identity.

The development of a ligase mediated PCR with potential for the differentiation of serovars within Leptospira interrogans.

A ligase mediated polymerase chain reaction (LMPCR) was developed to amplify between the repetitive element, IS1533, of Leptospira and adjacent chromosomally located Bg/II restriction endonuclease enzyme sites. To do this, complimentary oligonucleotide linkers designed to anneal together with an overhanging Bg/II end were ligated to Bg/II digested DNA from 35 leptospiral reference strains and field isolates. This ligated DNA was used as template for PCR with oligonucleotide primers specific for the linker and for the repetitive element IS1533. The resultant amplicon profile hybridised a 102 bp region derived from the terminus of IS1533 thus confirming that amplicons generated by LMPCR contained part of IS1533. The number of fragments generated containing IS1533 was significantly fewer than that generated by RFLP but the LMPCR method has the potential to use far less template DNA and be quicker than standard RFLP. Obvious and reproducible interserovar differences were demonstrated by LMPCR whereas for 20 of 21 L. hardjo-bovis isolates tested no intraserovar differences were observed. Of those serovars known to possess IS1533 homologues and tested here by LMPCR, each produced a unique amplicon profile which hybridised the IS1533 terminus probe. The limited heterogeneity amongst hardjo-bovis isolates is discussed as is the potential contribution of this method to diagnosis, differentiation and the phylogenetics of the Leptospires.

Deoxynucleotide sequence conservation of the endoflagellin subunit protein gene, flaB, within the genus Leptospira.

A Polymerase chain reaction was developed to amplify the entire open reading frame of flaB, the gene encoding the endoflagellin subunit protein. The 852 bp amplified products from 23 serovars of the genus Leptospira were subjected to restriction endonuclease analysis and the profiles correlated well with phylogenetic relationships between these serovars. The flaB deoxynucleotide sequences of L. hardjo-bovis, L. hardjo-prajitno and L. grippotyphosa were determined. The deduced primary amino acid sequences of each were highly conserved with only three amino acid residue differences observed. The deoxynucleotide sequences showed genetic drift with alternative bases in the third position of codons. The PCR product derived by amplification of flaB from L. grippotyphosa was cloned into the expression vector pGEX-2T and a recombinant FlaB fusion protein made. As predicted from the deduced amino acid sequences, the recombinant FlaB cross-reacted with heterologous antiserum derived from a rabbit infected with L. hardjo-bovis.