Finding Typhoid Mary: Identifying Latent Carriers of Salmonella enterica serovar Typhimurium
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Salmonella enterica serovar Typhimurium (S. Typhimurium) is an important human pathogen. The Centers for Disease Control and Prevention (CDC) estimates that 1,027,561 people become ill with nontyphoidal Salmonellosis annually, and S. Typhimurium is one of the most common disease causing serovars. Quantification of the true number of cases of salmonellosis is hampered by the presence of a carrier state. These carriers are animals and humans that carry the pathogens for a variable period of time without showing any clinical signs. One of the biggest barriers to controlling and preventing salmonellosis in a population is identification of these carriers. Identifying these latent carriers of chronic infections is vital to preventing such disease transmission and creating avenues for novel control and treatments. In my dissertation research, we developed a cell culture model to study latent Salmonella infections. By activating human monocytes with retinoic acid and vitamin D3, we were able to isolate Salmonella from such cells 45 days after inoculation. We subsequently used this model to identify genes that were upregulated in this chronic infection model. We found that aceA, a gene that codes for isocitrate lyase, is significantly upregulated on days 10 and 30 post infection. Isocitrate lyase is part of the glyloxylate cycle. Some bacterial species have developed a mechanism to utilize acetone as a carbon source to synthesize tricarboxylic acid (TCA) cycle intermediates. This anaplerotic reaction allows organisms to conserve carbon and use alternative carbon sources. This cycle is one way in which bacteria can adapt and survive in an intracellular environment. This intracellular survival is key to latent infections persisting within a host. It is biologically plausible that, in order to survive in a latent state, S. Typhimurium would up-regulate genes that would facilitate intracellular survival. After establishing the cell culture model, we tested the hypothesis that aceA is upregulated in latent infections of S. Typhimurium in a mouse model. We orally challenged mice that were resistant to Salmonella infection, collected their feces, and collected tissue specimens at several time points up to 135 days post-challenge. These samples were cultured and tested using quantitative polymerase chain reaction (qPCR). The qPCR results showed that tissue samples from inoculated mice had increased aceA expression 95 days after challenge. Finally, we examined whether aceA expression could be detected in cattle lymph node samples. Supra-mammary lymph nodes from 40 dairy cattle and mesenteric lymph nodes from 100 culled cattle were sampled and submitted for culture and qPCR. None of the supra-mammary lymph nodes were positive for Salmonella via culture or aceA qPCR; however, 11 mesenteric lymph nodes showed increased aceA expression in qPCR compared to 5 culture positive lymph nodes. Further research is necessary, but these results demonstrate some of the advantages of using genetic primers to identify latent Salmonella infections in clinically normal cattle. In addition, the assay may be able to differentiate between latent and active salmonellosis, and could be used to provide targeted drug delivery.
General Audience Abstract
Salmonella enterica serovar Typhimurium (S. Typhimurium) is an important human pathogen. Determining the true number of cases of salmonellosis is made more difficult because of the presence of a carrier state. These carriers are animals and humans that carry the pathogens for a variable period of time without showing any clinical signs. Identifying these latent carriers of chronic infections is vital to preventing such disease transmission and creating avenues for novel control and treatments. In my dissertation research, we looked at genetic markers from an offshoot of the TCA cycle, the glyoxylate pathway. We used these markers to test the hypothesis that these glyoxylate pathway genes would be upregulated in latent S. Typhimurium infections. Our research involved developing a cell culture model, then using the results from the cell culture model to inform a mouse model, and then a cattle lymph node diagnostic study. The cell culture model indicated that the gene for isocitrate lyase, aceA, is significantly upregulated compared to housekeeping genes. We found the presence of aceA in chronically infected mice, as well as cattle lymph node samples. Further research is necessary, but these results demonstrate some of the advantages of using genetic primers to identify latent Salmonella infections in clinically normal cattle.
- Doctoral Dissertations