Abstract
Salmonella enterica serovar Gallinarum biovars Pullorum (S. Pullorum) and Gallinarum (S. Gallinarum) can result in pullorum disease and fowl typhoid in avian species, respectively, and cause considerable economic losses in poultry in many developing countries. Conventional Salmonella serotyping is a time-consuming, labor-intensive and expensive process, and the two biovars cannot be distinguished using the traditional serological method. In this study, we developed a rapid and reliable one-step multiplex polymerase chain reaction (PCR) assay to simultaneously identify and discriminate the biovars Pullorum and Gallinarum. The multiplex PCR method focused on three specific genes, stn, I137_08605 and ratA. Based on bioinformatics analysis, we found that gene I137_08605 was present only in S. Pullorum and S. Gallinarum, and a region of difference in ratA was deleted only in S. Pullorum after comparison with that of S. Gallinarum and other Salmonella serovars. Three pairs of primers specific for the three genes were designed for the multiplex PCR system and their selectivity and sensitivity were determined. The multiplex PCR results showed that S. Pullorum and S. Gallinarum could be identified and discriminated accurately from all tested strains including 124 strains of various Salmonella serovars and 42 strains of different non-Salmonella pathogens. In addition, this multiplex PCR assay could detect a minimum genomic DNA concentration of 67.4 pg/μL, and 100 colony forming units. The efficiency of the multiplex PCR was evaluated by detecting natural-occurring Salmonella isolates from a chicken farm. The results demonstrated that the established multiplex PCR was able to identify S. Gallinarum and S. Pullorum individually, with results being consistent with traditional serotyping and biochemical testing. These results demonstrated that a highly accurate and simple biovar-specific multiplex PCR assay could be performed for the rapid identification and discrimination of Salmonella biovars Gallinarum and Pullorum, which will be useful, particularly under massive screening situations.
Highlights
Salmonellosis is a zoonotic disease, which can cause gastroenteritis, diarrhea, and systemic typhoid fever (McGhie et al, 2009)
Salmonella Gallinarum is the causative pathogen of fowl typhoid and results in variable morbidity and high mortality, which often leads to a severe septicemic disease occurring primarily in adult birds
Part of infected birds could get recovered from pullorum disease, and some adult birds may not present with any clinical disease symptoms, most birds develop a carrier state, and may become a repository for the pathogens, which may be transmitted vertically to newborn hatchlings and horizontally to other birds (Berchieri et al, 2001; Shivaprasad and Barrow, 2008)
Summary
Salmonellosis is a zoonotic disease, which can cause gastroenteritis, diarrhea, and systemic typhoid fever (McGhie et al, 2009). Part of infected birds could get recovered from pullorum disease, and some adult birds may not present with any clinical disease symptoms, most birds develop a carrier state, and may become a repository for the pathogens, which may be transmitted vertically to newborn hatchlings and horizontally to other birds (Berchieri et al, 2001; Shivaprasad and Barrow, 2008) These diseases have been wiped out from the commercial poultry industries in some developed countries, they are widespread in developing countries such as Asia, Africa, and Central and South America, and have caused enormous economic losses (Jones et al, 2001; Kang et al, 2011)
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