Differential identification of Salmonella enterica serovar Gallinarum biovars Gallinarum and Pullorum and the biovar Gallinarum live vaccine strain 9R.
Salmonella enterica serovar Gallinarum biovar Gallinarum causes fowl typhoid in chickens and has been of economic importance to the chicken industry in many countries. The biovar Gallinarum live vaccine strain 9R (SG 9R) has been used to control fowl typhoid in many areas where the disease is endemic. Therefore, a definitive diagnosis of this disease may require differentiation of wild-type field isolates of biovar Gallinarum from the live vaccine strain SG 9R. Here, we report the development of a triplex polymerase chain reaction (PCR) assay to differentially identify serovar Gallinarum biovars Gallinarum and Pullorum and SG 9R. Sequences specific to SG 9R, which are absent or highly divergent in the fully sequenced biovar Gallinarum strain 287/91, were identified by constructing the suppression subtractive hybridization (SSH) library. A total of 565 nonredundant inserts were obtained from successfully sequenced SSH clones (718 clones). Sequences of 14 inserts were unique to SG 9R, but single nucleotide polymorphisms (SNPs) found in another insert (9R22C9) were more useful for strain discrimination. A new PCR primer set was designed to target SNP regions of the insert and was integrated into a duplex PCR assay developed previously (Kang et al., 2011). Boiled lysates of 138 reference and field strains of Salmonella and other related Gram-negative bacteria were tested to validate the triplex PCR assay. All strains of biovars Gallinarum (n=53) and Pullorum (n=21) and SG 9R (n=7) tested were differentially identified, whereas the other strains (n=57) were PCR negative. This triplex PCR assay will be very useful for rapid differential diagnoses of fowl typhoid and pullorum disease in veterinary laboratories.
Animal, Plant and Fisheries Quarantine and Inspection Agency, Ministry for Food, Agriculture, Forestry and Fisheries, Anyang, Gyeonggi, Republic of Korea. kangmskr@korea.kr
Min-Su
MS
Kang
Yong-Kuk
YK
Kwon
Hye-Ryoung
HR
Kim
Jae-Young
JY
Oh
Mi-Jin
MJ
Kim
Byung-Ki
BK
An
Eun-Gyeong
EG
Shin
Jun-Hun
JH
Kwon
Choi-Kyu
CK
Park
D000818:Animals; D001483:Base Sequence; D002645:Chickens; D017931:DNA Primers / Q000235:genetics; D008969:Molecular Sequence Data; D058889:Molecular Typing / Q000379:methods*; D016133:Polymerase Chain Reaction / Q000592:standards / Q000662:veterinary*; D020641:Polymorphism, Single Nucleotide / Q000235:genetics; D011201:Poultry Diseases / Q000175:diagnosis / Q000382:microbiology*; D015203:Reproducibility of Results; D012481:Salmonella Infections, Animal / Q000175:diagnosis / Q000382:microbiology*; D019779:Salmonella enterica / Q000235:genetics*; D016415:Sequence Alignment; D013045:Species Specificity; D014613:Vaccines, Attenuated / Q000235:genetics
D017931:DNA Primers; D014613:Vaccines, Attenuated