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The genus Listeria consists of six species: L. monocytogenes, L. innocua, L. welshimeri, L. ivanovii, L. seeligeri and L. grayi. Two of the species, L. monocytogenes and L. ivanovii are pathogenic. The heterogeneity of remaining species, previously assumed to be nonpathogenic, regarding their capability of acquiring virulence-associated genes may reflect their potential ability to be causative agents of diseases, especially in immunocompromised mannals. Virulence determinants involved in environmental tolerance, adhesion and invasion of eukaryotic cells and intracellular life function interactively. The virulence genes are mostly organized into discrete genetic units known as pathogenicity islands (PAIs), among which Listeria pathogenicity island 1 (LIPI-1) and island 2 (LIPI-2) are the most important. During the evolution of pathogenicity, a common ancestor bearing PAIs gave rise to the currently prevailing typical strains of six species through horizontal transfer of virulence determinants or by events such as recombination and natural selection. Bacteriophages, transposons and plasmids might play critical roles in these processes as the executants. Compred to pathogenic species, the nonpathogenic species lost LIPI-1 (L. innocua, L. welshimeri and L. grayi) or harbored corrupted LIPI-1 (L. innocua, L. welshimeri). Some types of natural atypical Listeria strains such as nonhemolytic L. seeligeri and hemolytic L. innocua, although complicating taxonomic identification, should contribute fruitful insights into the evolution events of pathogenicity underlying the phylogeny of the genus Listeria.

Vibrio parahaemolyticus is a gram-negative, halophilic bacterium that inhabits the marine and estuarine environments. It is an important human pathogen causing gastroenteritis when raw or partially-cooked seafoods are consumed. Its pathogenicity is believed to be related to hemolysins such as thermostable direct hemolysin (TDH), TDH-related hemolysin (TRH) and thermolabile hemolysin (TLH). PCR method was used to examine three different hemolysin genes in isolates from clinical and seafood samples in Zhejiang province. The tlh gene was found in all isolates. The tdh gene was positive in all eleven clinical strains but only in one out of a total of 42 seafood isolates. The Kanagawa phenomenon was positive for all tdh-positive isolates. None of the isolates was positive for the trh gene. The urease test was negative for all isolates. Thus, it was assumed that the urease gene could be linked with trh gene. Further research is required to examine the relationship between low prevalence of the major virulence factor TDH and the high incidence of foodborne V. parahaemolyticus infections,and its pathogenesis.

Homologous recombination was utilized for construction of a recombinant strain of L. monocytogenes carrying a gene from the Newcastle diseases virus by insertional mutation targeting its listeriolysin O gene (hly). The gene encoding fusion protein of the Newcastle disease virus (NDV-F) was used as the model heterologous gene. The F gene was inserted into hly downstream to its promoter and signal sequence by overlapping extension polymerase chain reaction, which was then subcloned into the shuttle plasmid pKSV7 for allelic exchange with L. monocytogenes chromosome. PCR amplification of the target genes indicated insertion of the F gene into the chromosome DNA of L. monocytogenes. RT-PCR showed transcription of F gene from the recombinant L. monocytogenes strain. Comparisons were then made between the recombinant strain and its wild parent strain in terms of the hemolytic activity, adhesion and invasiveness to cultured HeLa cells, virulence to mice and chicken embryos, and growth kinetics in broth medium as well as its stability upon repeated subculturing and serial passages in mice. The recombinant L. monocytogenes lost its hemolytic activity on the blood agar and had no hemolytic titer from its culture supernatants as compared with the titer of 24 in the supernatant from the wild parent strain. The recombinant strain also had lower adhesiveness (P > 0.05) and significantly lower relative invasiveness to the HeLa cells than its wild type strain (P < 0.05). Such insertional mutation resulted in reduced virulence, about 3.7 logs and 6.5 logs less than its parent strain L. monocytogenes 10403S as shown by the 50% lethal dose assays in the mouse and chicken embryonated egg models respectively. The recombinant strain was relatively stable as shown by amplification of the target gene NDV-F from its genomic DNA after subculturing in BHI broth or in mice for 5 times.

Effects of plasmid type and insertion sequence on in vitro and in vivo multiplication and invasiveness of attenuated Salmonella typhimurium were examined following transformation of the bacteria with eukaryotic expression plasmids pcDNA3 and pCI with or without heterologous gene (Newcastle disease virus F gene). Exogenous plasmids had negative impacts on the replication or invasiveness of the attenuated S. typhimurium in LB broth and/or HeLa cell monolayers as well as on its survival in live chicks. The plasmid pCI had more significant effects than pcDNA3. Introduction of heterologous gene into the plasmids not only exhibited additional negative influence on the host strain but also on their own stability therein. All these results suggest that full consideration should be given to the types of plasmids and their stability within the host strain as well as to their effects on replication and invasiveness of attenuated bacteria as the DNA vaccine delivery vector for improved immune protection.