• Korean Journal of Veterinary Service
• /
• v.33 no.4
• /
• pp.341-344
• /
• 2010

Poultry and poultry products have been implicated as a major source of Salmonella infection in human, and infection due to Salmonella serotypes continue to be a major health problem. The presence of two virulence genes, invA and spvC, in 34 Salmonella isolates obtained from duck farms was investigated. All isolates contained the invA gene, and spvC gene was found in 20 (58.8%) of 34 Salmonella isolates : S. Typhimurium (n=8), S. Fyris (n=5), S. Enteritidis (n=3), S. Typhimurium var. copenhagen (n=1), S. Haardt (n=1) and S. Mbandaka (n=1). This study showed the presence of the spvC gene was widely distributed in between different Salmonella enterica isolates.

• Journal of Microbiology and Biotechnology
• /
• v.15 no.6
• /
• pp.1353-1358
• /
• 2005

A rapid and quantitative real-time PCR was developed to target the invasion A (invA) gene of Salmonella spp. We developed quantitative standard curves based on plasmids containing the invA gene. Based on these curves, we detected Salmonella spp. in artificially contaminated buffered peptone water (BPW) and milk samples. We were able to determine the invA gene copy number per ml of food samples, with the minimum detection limit of $4.1{\times}10^{3}$ copies/ml of BPW and $3.3{\times}10^{3}$ copies/ml of milk. When applied directly to detect and quantify Salmonella spp. in BPW and milk, the present real-time PCR assay was as sensitive as the plate count method; however, copy numbers were one to two logs higher than the colony-forming units obtained by the plate count methods. In the present work, the real-time PCR assay was shown to significantly reduce the total time necessary for the detection of Salmonella spp. in foods and to provide an important model for other foodborne pathogens.

• Microbiology and Biotechnology Letters
• /
• v.40 no.1
• /
• pp.70-75
• /
• 2012

In Salmonella enterica, many genes encoded within Salmonella pathogenicity islands (SPI) 1 and 2 are required to cause a range of diseases in a variety of hosts. The SPI1-encoded regulator HilD activates both the SPI1 and 2 genes at different times during growth in Luria-Bertani (LB) media. In this study, the expression levels of hilD during growth in LB were investigated. The data suggest that hilD expression is induced in the early stationary phase and decreases in the late stationary phase, when sseA, an SPI2 gene, is maximally expressed. However, HilD could act as an activator of sseA expression in the late stationary phase despite being present at low levels. SseA expression was investigated in SPI1 regulator mutant strains, hilA, hilD and invF mutants. As expected, hilD mutation decreased sseA expression. However, we found that invF mutation caused a 1.5-fold increase in sseA expression in not only LB but also M9 minimal media, which is thought to resemble an intracellular environment. InvF overexpression restored sseA expression to wild-type levels in an invF mutant but did not cause an additional reduction in sseA expression. These results suggest that SPI1 controls SPI2 expression either positively or negatively.

• Journal of Food Hygiene and Safety
• /
• v.22 no.4
• /
• pp.248-256
• /
• 2007

In this study, the competitive polymerase chain reaction (cPCR) was used to develop a direct enumeration method of Salmonella spp. in pork meat. After comparing three DNA extraction methods, the modified guanidine thiocyanate-phenol-chloroform method was chosen for Salmonella DNA extraction in artificially inoculated pork meat. The previously reported 284-bp invA gene (Rahn et al. Mol. Cell. Probes 1992) was tested for specificity, and 57 Salmonella strains and 24 non-Salmonella strains were evaluated. All Salmonella strains tested were invA positive, and all non-Salmonella strains produced no false positive amplification products. The detection limit achieved was as low as 1,460 colony-forming units (cfu) per 0.1g of pork meat. For cPCR, the invA gene, which features a 82 bp-deletion, was cloned in the pGEM-4Z vector. A known amount of competitor DNA, which has the same primer binding sites, was co-amplified with Salmonella chromosomal DNA from the artificially inoculated pork meat. The cell-number determined by cPCR was approximately equal to the cfu from the most probable number (MPN) method. Finally, the whole procedure took only 5 hr.

• Food Science and Biotechnology
• /
• v.16 no.5
• /
• pp.778-782
• /
• 2007

This study assessed the effects of the antimicrobial substances sulforaphane, grapefruit seed extracts (GSE), and reuterin on the expression of Salmonella HilA and InvF virulence gene using a LacZY assay (${\beta}$-galactosidase assay) with hilA:lacZY and invF:lacZY fusion strains of Salmonella typhimurium SL1344. Salmonella was grown for 8 hr at $37^{\circ}C$ in the presence of diluted antimicrobial substances ($2\;{\mu}g/mL$ sulforaphane, $20\{\mu}g/mL$ GSE, and 0.26 mM reuterin) at concentrations that did not inhibit the cellular growth of Salmonella. Sulforaphane inhibited the expression of HilA and InvF by 50-90 and 20-80%, respectively. GSE also inhibited the expression of both genes, but to a lesser degree. Among the 3 antimicrobial substances, reuterin showed the least inhibition, which was abolished after 3-4 hr. None of the antimicrobial substances inhibited the ${\beta}$-galactosidase enzyme activity of S. typhimurium. The assay used in this study represents a very sensitive method for screening bioactive substances that inhibit the expression of virulence genes in Salmonella.

• Journal of Veterinary Science
• /
• v.24 no.6
• /
• pp.82.1-82.12
• /
• 2023

Background: The current conventional serotyping based on antigen-antisera agglutination could not provide a better understanding of the potential pathogenicity of Salmonella enterica subsp. enterica serovar Brancaster. Surveillance data from Malaysian poultry farms indicated an increase in its presence over the years. Objective: This study aims to investigate the virulence determinants and antimicrobial resistance in S. Brancaster isolated from chickens in Malaysia. Methods: One hundred strains of archived S. Brancaster isolated from chicken cloacal swabs and raw chicken meat from 2017 to 2022 were studied. Two sets of multiplex polymerase chain reaction (PCR) were conducted to identify eight virulence genes associated with pathogenicity in Salmonella (invasion protein gene [invA], Salmonella invasion protein gene [sipB], Salmonella-induced filament gene [sifA], cytolethal-distending toxin B gene [cdtB], Salmonella iron transporter gene [sitC], Salmonella pathogenicity islands gene [spiA], Salmonella plasmid virulence gene [spvB], and inositol phosphate phosphatase gene [sopB]). Antimicrobial susceptibility assessment was conducted by disc diffusion method on nine selected antibiotics for the S. Brancaster isolates. S. Brancaster, with the phenotypic ACSSuT-resistance pattern (ampicillin, chloramphenicol, streptomycin, sulphonamides, and tetracycline), was subjected to PCR to detect the corresponding resistance gene(s). Results: Virulence genes detected in S. Brancaster in this study were invA, sitC, spiA, sipB, sopB, sifA, cdtB, and spvB. A total of 36 antibiogram patterns of S. Brancaster with a high level of multidrug resistance were observed, with ampicillin exhibiting the highest resistance. Over a third of the isolates displayed ACSSuT-resistance, and seven resistance genes (β-lactamase temoneira [bla_TEM], florfenicol/chloramphenicol resistance gene [floR], streptomycin resistance gene [strA], aminoglycoside nucleotidyltransferase gene [ant(3")-Ia], sulfonamides resistance gene [sul-1, sul-2], and tetracycline resistance gene [tetA]) were detected. Conclusion: Multidrug-resistant S. Brancaster from chickens harbored an array of virulence-associated genes similar to other clinically significant and invasive non-typhoidal Salmonella serovars, placing it as another significant foodborne zoonosis.

• Korean Journal of Veterinary Service
• /
• v.26 no.1
• /
• pp.39-50
• /
• 2003

Non-typhoidal Salmonella serovars remain a potential threat to human health and many animals including beef cattle, broiler chickens, and pigs which possible sources of non-typhoidal salmonellosis in human. In this study, the cecal contents of slaughtered pigs were examined for Salmonella serovar prevalence. The characteristics of the isolates, including antimicrobial resistance patterns and virulence genes, were studied along with the reference strain S typhimurium ATCC 13311. Out of 640 sample, 137 Salmonella(21.4%) were isolated and their serovar were identified S typhimurium 83 strains(60.6%), S agona 10 strains(7.3%), S schwarzengrund 4 strains(2.9%), S derby 4 strains(2.9%), S ayinde 1 strains(0.7%), and untypable 35 strains(25.5%). All 83 S typhimurium strains(100%) were multi-drug resistance to at least 7 antibiotics, and 20 strains(24.1%) of 83 isolates were R-type ACSSuT. Examination of virulent gene by PCR revealed that 73 S typimurium field isolates(88%) have a invA gene and 24 strains(28.9%) have a spvC gene. Consequently, S typhimurium infection in slaughtered pigs was relatively to appear high prevalence in their herds which suggested that it should be necessary for herd health monitoring and surveillance.

• Animal Bioscience
• /
• v.36 no.4
• /
• pp.654-670
• /
• 2023

Objective: This study aimed to develop loop-mediated isothermal amplification (LAMP) combined with lateral flow dipstick (LFD) and compare it with LAMP-AGE, polymerase chain reaction (PCR), and standard Salmonella culture as reference methods for detecting Salmonella contamination in animal products and animal production environmental samples. Methods: The SalInvA01 primer, derived from the InvA gene and designed as a new probe for LFD detection, was used in developing this study. Adjusting for optimal conditions by temperature, time, and reagent concentration includes evaluating the specificity and limit of detection. The sampling of 120 animal product samples and 350 animal production environmental samples was determined by LAMP-LFD, comparing LAMP-AGE, PCR, and the culture method. Results: Salmonella was amplified using optimal conditions for the LAMP reaction and a DNA probe for LFD at 63℃ for 60 minutes. The specificity test revealed no cross-reactivity with other microorganisms. The limit of detection of LAMP-LFD in pure culture was 3×10² CFU/mL (6 CFU/reaction) and 9.01 pg/μL in genomic DNA. The limit of detection of the LAMP-LFD using artificially inoculated in minced chicken samples with 5 hours of pre-enrichment was 3.4×10⁴ CFU/mL (680 CFU/reaction). For 120 animal product samples, Salmonella was detected by the culture method, LAMP-LFD, LAMP-AGE, and PCR in 10/120 (8.3%). In three hundred fifty animal production environmental samples, Salmonella was detected in 91/350 (26%) by the culture method, equivalent to the detection rates of LAMP-LFD and LAMP-AGE, while PCR achieved 86/350 (24.6%). When comparing sensitivity, specificity, positive predictive value, and accuracy, LAMP-LFD showed the best results at 100%, 95.7%, 86.3%, and 96.6%, respectively. For Kappa index of LAMP-LFD, indicated nearly perfect agreement with culture method. Conclusion: The LAMP-LFD Salmonella detection, which used InvA gene, was highly specific, sensitive, and convenient for identifying Salmonella. Furthermore, this method could be used for Salmonella monitoring and primary screening in animal products and animal production environmental samples.

• Journal of Microbiology and Biotechnology
• /
• v.23 no.12
• /
• pp.1664-1672
• /
• 2013

In Salmonella enterica serovar Typhimurium, many genes encoded within Salmonella pathogenicity island 1 (SPI1) are required to induce intestinal/diarrheal disease. In this study, we compared the expression of four SPI1 genes (hilA, invF, prgH, and sipC) under shaking and standing culture conditions and found that the expression of these genes was highest during the transition from the exponential to stationary phase under shaking conditions. To identify regulators associated with the stationary phase-dependent activation of SPI1, the effects of selected regulatory genes, including relA/spoT (ppGpp), luxS, ihfB, hfq, and arcA, on the expression of hilA and invF were compared under shaking conditions. Mutations in the hfq and arcA genes caused a reduction in hilA and invF expression (more than 2-fold) in the early stationary phase only, whereas the lack of ppGpp and IHF decreased hilA and invF gene expression during the entire stationary phase. We also found that hfq and arcA mutations caused a reduction of hilD expression upon entry into the stationary phase under shaking culture conditions. Taken together, these results suggest that Hfq and ArcA regulate the hilD promoter, causing an accumulation of HilD, which can trigger a stationary phase-dependent activation of SPI1 genes under shaking culture conditions.

• Journal of Microbiology and Biotechnology
• /
• v.17 no.10
• /
• pp.1616-1621
• /
• 2007

We have established a SYBR Green-based realtime PCR method using AnyDirect solution, which enhances PCR from whole blood, for direct amplification of the virA gene of Shigella flexneri and the invA gene of Salmonella typhimurium from human feces without prior DNA purification. When we compared the efficiency of conventional or realtime PCR amplification of the virA and invA genes from the supernatant of boiled feces supplemented with S. flexneri and S. typhimurium in the presence or absence of AnyDirect solution, amplification products were detected only in reactions to which AnyDirect solution had been added. The detection limit of real-time PCR was $1{\times}10^4\;CFU/g$ feces for S. flexneri and $2{\times}10^4\;CFU/g$ feces for S. typhimurium; this sensitivity level was comparable to other studies. Our real-time PCR assay with AnyDirect solution is simple, rapid, sensitive, and specific, and allows simultaneous detection of S. flexneri and S. typhimurium directly from fecal samples without prior DNA purification.