Devendra H. Shah

An Allele-Specific PCR Assay for the Rapid and Serotype-Specific Detection of Salmonella Pullorum

Abstract Salmonella serovar Pullorum is a causative agent of pullorum disease (PD) in poultry and is responsible for severe economic losses to the poultry industry in many parts of the world. A definitive detection of Pullorum requires culture followed by serotyping and biochemical identification, a process that is tedious and takes several weeks to accomplish. We have developed a rapid allele-specific polymerase chain reaction (PCR) method based on the nucleotide polymorphism in rfbS gene sequence for the serotype-specific detection of Pullorum and its differentiation from the closely related Gallinarum. The specificity of this PCR assay was tested using DNA samples from Pullorum (n = 13), Salmonella serotypes other than Pullorum (n = 19), and closely related non-Salmonella organisms (n = 5). The PCR assay was highly serotype-specific as the PCR amplicon of 147 base pairs was observed only in the case of Pullorum, while all the other DNA samples tested PCR negative. A definitive identification of Pullorum cultures was possible in less than 3 hr. As little as 100 pg of SP DNA was detected. This allele-specific PCR method is highly specific as well as sensitive and may be an effective molecular tool in the rapid and serotype-specific detection of Pullorum and differentiation from other Salmonella species.

Cloning and expression of 51-kDa antigenic protein of Neorickettsia risticii NR-JA1.

Abstract: Neorickettsia (Ehrlichia) risticii is a causative agent of acute diarrheal syndrome in horses, commonly known as Potomac horse fever. Korean isolate of N. risticii NR‐JA1 was cultivated in mouse macrophage cell line P388D1. A complete ORF of p51 antigenic protein gene was amplified and cloned into pQE32 and pcDNA3.1 vectors and the resultant clones were named as pQE32/Nr‐51 and pcDNA3.1/Nr‐51, respectively. Recombinant p51 (rp51) protein antigen was expressed in E. coli (pQE32/Nr‐51) and cos‐7 cell line (pcDNA3.1/Nr‐51). The rp51 protein showed immunoreactivity with anti‐ mouse p51 antibodies. BALB/c mice were inoculated with recombinant plasmid DNA (pcDNA3.1/Nr‐51). The serum samples collected from these BALB/c mice showed IgG ELISA titers of 1:128. In a Western immunoblot assay, these serum samples showed a strong reactivity to rp51 expressed in cos‐7 cell line transfected with pcDNA3.1/Nr‐51. The results of this preliminary indicate that N. risticii p51 protein is an immmuno‐dominant antigen and may be a good target for the development of serological or a molecular diagnostic test and possibly an improved recombinant DNA based vaccine against Potomac horse fever.