Name: an allelotyping pcr for identifying salmonella enterica serovars enteritidis, hadar, heidelberg, and typhimurium
Uploaded: 2011-07-22T13:00:00
Duration: 12 min 18 s
Description: Watch this Scientific Journal Video about An Allelotyping PCR for Identifying Salmonella enterica serovars Enteritidis, Hadar, Heidelberg, and Typhimurium at JoVE.com

This work has been supported by USDA Grants 1999-35212-8680, 2005-01378, and 2010-04288-01, USDA Formula Funds and the State of Georgia&#x2019;s Veterinary Medicine Agricultural Research Grant. The protocol described in this publication was developed for use by undergraduates as part of directed research courses: MIBO 4900L, GENE 4960H, BCMB 4960L, and BIOL 4960H at the University of Georgia and used by the students on several molecular epidemiology research projects in the Maurer laboratory. We wish to thank the many undergraduate students who have performed research in Maurer and Lee laboratories, and our department chair, John Glisson for supporting our efforts to integrate undergraduate instruction with research.

<ol>
	<li>Dauga, C., Zabrovskaia, A., Grimont, P. A. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Restriction+fragment+length+polymorphism+analysis+of+some+flagellin+genes+of+Salmonella+enterica.">Restriction fragment length polymorphism analysis of some flagellin genes of Salmonella enterica.</a> J. Clin. Microbiol. 36, 2835-2843 .</li><li>Hong, Y., Liu, T., Lee, M. D., Hofacre, C. L., Maier, M., White, D. G., Ayers, S., Wang, L., Berghaus, R., Maurer, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+rapid+screen+of+broth+enrichments+for+Salmonella+enterica+serovars+Enteritidis,+Hadar,+Heidelberg,+and+Typhimurium+using+an+allelotyping+multiplex+PCR+that+targets+O+and+H+antigen+alleles.">A rapid screen of broth enrichments for Salmonella enterica serovars Enteritidis, Hadar, Heidelberg, and Typhimurium using an allelotyping multiplex PCR that targets O and H antigen alleles.</a> J. Food Prot. 72, 2198-2201 (2009).</li><li>Hong, Y., Liu, T., Lee, H. o. f. a. c. r. e., Maier, C. L., White, M., Ayers, D. G., Wang, S., Berghaus, L., R, M. a. u. r. e. r. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=J.Rapid+screening+of+Salmonella+enterica+serovars+Enteritidis,+Hadar,+Heidelberg+and+Typhimurium+using+a+serologically-correlative+allelotyping+PCR+targeting+the+O+and+H+antigen+alleles.">J.Rapid screening of Salmonella enterica serovars Enteritidis, Hadar, Heidelberg and Typhimurium using a serologically-correlative allelotyping PCR targeting the O and H antigen alleles.</a> BMC Microbiol. , (2008).</li><li>Liu, T., Liljebjelke, K., Bartlett, E., Hofacre, C. L., Sanchez, S., Maurer, J. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Application+of+nested+PCR+to+detection+of+Salmonella+in+poultry+environments.">Application of nested PCR to detection of Salmonella in poultry environments.</a> J. Food Prot. 65, 1227-1232 (2002).</li><li>Maurer, J. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rapid+detection+and+limitation+of+molecular+techniques.">Rapid detection and limitation of molecular techniques.</a> Ann. Rev. Food Sci. Tech. 2, 259-279 (2011).</li><li>Moscoso, H., Alvarado, I., Hofacre, C. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Molecular+analysis+of+infectious+bursal+disease+virus+from+bursal+tissues+collected+on+FTA+filter+paper.">Molecular analysis of infectious bursal disease virus from bursal tissues collected on FTA filter paper.</a> Avian Dis. 50, 391-396 (2006).</li><li>Sambrook, J., Fritsch, E. F., Maniatis, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Molecular cloning: a laboratory. , (1989).</li><li>Wittwer, C. T., Fillmore, G. C., Hillyard, D. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Automated+polymerase+chain+reaction+capillary+tubes+with+hot+air.">Automated polymerase chain reaction capillary tubes with hot air.</a> Nucleic Acids Res. 17, 4353-4357 (1989).</li></ol>

No conflicts of interest declared.

Most commercially available PCR tests for the detection of Salmonella targets genes unique to the genus (ex. invA). However, not all Salmonella are equal in their ability to cause disease in humans or prevalence in animal populations. Early recognition of antigenic differences in Salmonella LPS O-antigen and flagellinH1 and H2 antigens has lead to the differentiation of Salmonella into over 2,500 serovars based on these antigenic differences. There are 46 distinct O antigens and 86 distinct flagellin (H) antigens identified in the genus Salmonella. Salmonella may possess one (H1) or two different (H1&amp;H2) flagellins. The different combination of O, H1, and H2 antigens account for the 2,500 Salmonella serovars recognized today. A serovar is assigned based on the antigenic formula derived from the identification of the individual O and H antigens. The antigenic formula is written as O: H1: H2; and where "-", refers to the absence of H1 or H2 flagellin and "NT" (non-typeable) for Salmonella negative for the O-antigen. For example, the serovar Typhimurium is assigned to a S. enterica isolate with the antigenic formula B: i: 1,2 while Enteritidis is given to an isolate with the formula D1: g,m: -. This antigenic variation in the Salmonella population is the outward manifestation of differences at the nucleotide level that can therefore be easily exploited by PCR. 
We have identified the genetic differences associated with specific O and H alleles to develop an allelotyping PCR for identifying S. enterica serovars: Enteritidis, Hadar, Heidelberg, and Typhimurium (3). The correct assignment and reporting of Salmonella serovar requires testing for all alleles associated with O: H1: H2 antigenic formulas for Enteritidis (D1: g,m: -), Hadar (C2: z10: e,n,x), Heidelberg (B: r: 1,2), and Typhimurium (B: i: 1,2). Without knowledge of O allele or antigen, the finding of the H1 g,m allele alone does not necessarily identify the Salmonella isolate as Enteritidis as other Salmonella serovars: Agona, California, and Montevideo, also possess this same allele. While the allelotyping PCR was originally designed to detect the fore mentioned Salmonella serovars, this test can identify an additional 19 serovars (Table 3). Our allelotyping PCR was originally designed to detect O and H alleles. In practice, it has become more practical and cost effective for us to identify the O-antigen by slide agglutination test, using O-specific antisera, rather than performing the O allelotyping PCR when working with isolated Salmonella cultures. However, we do recommend using the O allelotyping PCR if your lab uses this PCR as a screen (2) or for typing Salmonella isolates submitted on FTA cards (6), and include a Salmonella-specific PCR with the first screen of samples (4). Limit the allelotyping PCRs to only those samples that are identified as Salmonella by PCR or biochemical/antigenic tests.
The protocol described in this article has been optimized for use with the Idaho Technology Rapidcycler, a hot-air thermocycler that allows one to scale down reaction volumes and runs reaction conditions in less time than many heating block thermocyclers. To adapt this protocol for use with a heating block thermocycler may require optimizing reaction conditions empirically by lowering/raising annealing temperatures; adjusting primer concentrations; or adjusting MgCl2 concentrations. For example, we have had to adapt the protocol for the MJ Research PTC-200 thermocycler as follows. Working with the same reaction volumes described in Table 1, the working stock concentrationof the i primer set was diluted to 2.5 &mu;M, the annealing temperature was lowered from 55&deg;C to 45&deg;C, and incubation times at the denaturation, annealing and extension steps was lengthened to 20 s for the i/g, m allelotyping PCR. Having the appropriate positive and negative controls are essential in the initial start up and optimization for any PCR, including published protocols. We recommend acquiring known Salmonella serovars, specifically Anatum (E1: e,h: 1,6), Enteritidis (D1: g,m: -), Hadar (C2: z10: e,n,x), Heidelberg (B: r: 1,2), Montevideo (C1: g,m,s: 1,2,7) and Typhimurium (B: i: 1,2); and a laboratory Escherichia coli K12 strain (DH5&alpha;, LE393, JM109, etc.) as positive and negative controls respectively for the allelotyping PCR tests described in this article. Several of these Salmonella serovars will serve as either positive or negative controls, depending on which allele is tested.
Certain precautions and guidelines need to be followed while performing this and any other diagnostic PCR. First, the physical separation of template preparation, PCR set-up, and gel electrophoresis is critical in preventing possible PCR carry-over contamination and erroneous reporting of false-positives. In addition, the inclusion of appropriate controls is necessary in any routine PCR as these controls identify problems as they arise and assist in the interpretation of results (5). Most importantly, consistency is essential, especially with regards to the electrophoretic conditions for amplicon (PCR product) detection andestimation of amplicon size. To illustrate this point, we purposely suspended electrophoresis earlier than intended in Figure 1A. The molecular weight standards (lanes 1 and 13) and positive control (lane 2) are not sufficiently separated to accurately estimate sizes or observe separation of DNA fragments where there are small differences (&tilde;50 bp) in sizes. Adequate separation of DNA fragments, especially the molecular weight standards, is essential as reporting positives is dependent on the accurate estimation of amplicon size and distinguishing "true positive" from non-specific amplicons that are sometimes observed in the daily running of any PCR (5). For example, there is a non-specific amplicon in Figure 1B, lane 7 (&tilde;700 bp in size). Had electrophoresis been discontinued too early, when the dye front was only at the half way point in the agarose gel, there would be little differentiation between 500 bp and the 700 bp DNA fragments. Therefore, sample in lane 7 would have been erroneously reported as positive for both i and g,m alleles. 
Finally, one needs to recognize the limitations associated with any test. Several of the H1/H2 allelotyping PCR do not have the discriminatory power to discern subtle genetic differences in alleles belonging to the H2 1,2; 1,5; 1,6; 1,7 antigen complex; e,n,x/e,n,z15 antigen complex and the H1 G antigen complex, which includes the g,m allele. One or two amino acid changes account for the different epitopes present in these flagellar antigens. It was therefore difficult for us to design primers that could exploit these sometimes single base pair differences in the flagellin gene sequence (3). For example, Salmonella serovars Dublin (D1: g,p: -) and Berta (D1: f,g,t: -) are also PCR positive with our g,mallelotyping primers. The H2 allelotyping primers cannot distinguish Typhimurium (B: i : 1,2) from Lagos (B: i: 1,5), Heidelberg (B: r: 1,2) from Bradford (B: r: 1,5), Winneba (B: r: 1,6), or Remo ( B: r: 1,7), or Hadar (C2: z10: e,n,x) from Glostrup (C2: z10: e,n,z15). While the likelihood of encountering some of these serovars: Lagos, Bradford, Winneba, Remo or Glustrup is remote, it is a possibility and requires either providing disclaimer on the tests' limitation or another confirmatory test. 
In conclusion, this PCR-based serotyping rapidly identifies S. enterica serovars Enteritidis, Hadar, Heidelberg and Typhimurium, and O, H1, and H2 alleles associated with 19 additional serovars. This assay is a rapid, cost-effective alternative to traditional microbiological approach to serotyping Salmonella.

<table border="1">
 <tbody>
 <tr>
 <th>Material Name</th>
 <th>Type</th>
 <th>Company</th>
 <th>Catalogue Number</th>
 <th>Comment</th>
 </tr>
 <tr>
 <td>Luria Bertani</td>
 <td>&nbsp;</td>
 <td>Fisher</td>
 <td>&nbsp;</td>
 <td>(or any comparable source)</td>
 </tr>
 <tr>
 <td>Microfuge tubes</td>
 <td>1.5 ml capacity</td>
 <td>Fisher</td>
 <td>&nbsp;</td>
 <td>(or any comparable source)</td>
 </tr>
 <tr>
 <td>Ethanol</td>
 <td>200 proof</td>
 <td>Fisher</td>
 <td>&nbsp;</td>
 <td>(or any comparable source)</td>
 </tr>
 <tr>
 <td>dH2O</td>
 <td>Molecular Biology Grade</td>
 <td>Sigma</td>
 <td>&nbsp;</td>
 <td>(or any comparable source; PCR only)</td>
 </tr>
 <tr>
 <td>10 x PCR Buffer:</td>
 <td>20 mM MgCl2</td>
 <td>Idaho Technology</td>
 <td>&nbsp;</td>
 <td>(buffer comes with BSA, Ficoll and Tartrazine)</td>
 </tr>
 <tr>
 <td>&nbsp;</td>
 <td>30 mM MgCl2</td>
 <td>&nbsp;</td>
 <td>&nbsp;</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>&nbsp;</td>
 <td>40 mM MgCl2</td>
 <td>&nbsp;</td>
 <td>&nbsp;</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>PCR primers</td>
 <td>&nbsp;</td>
 <td>&nbsp;</td>
 <td>&nbsp;</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>DMSO</td>
 <td>&nbsp;</td>
 <td>&nbsp;</td>
 <td>&nbsp;</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>dNTP</td>
 <td>&nbsp;</td>
 <td>Roche</td>
 <td>&nbsp;</td>
 <td>(or any comparable source)</td>
 </tr>
 <tr>
 <td>Taq DNA Polymerase</td>
 <td>&nbsp;</td>
 <td>Denville</td>
 <td>&nbsp;</td>
 <td>(or any comparable source)</td>
 </tr>
 <tr>
 <td>Capillary pipettes</td>
 <td>10 &mu;l volume</td>
 <td>Idaho Technology</td>
 <td>&nbsp;</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Rapid Cycler</td>
 <td>&nbsp;</td>
 <td>Idaho Technology</td>
 <td>&nbsp;</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Agarose</td>
 <td>Low EEO; Molecular Biology Grade</td>
 <td>BioRad</td>
 <td>&nbsp;</td>
 <td>(or any comparable source)</td>
 </tr>
 <tr>
 <td>50 X TAE Buffer or 5X TBE Buffer</td>
 <td>&nbsp;</td>
 <td>Fisher</td>
 <td>&nbsp;</td>
 <td>(or any comparable source)</td>
 </tr>
 <tr>
 <td>Ethidium bromide</td>
 <td>&nbsp;</td>
 <td>BioRad</td>
 <td>&nbsp;</td>
 <td>(or any comparable source)</td>
 </tr>
 <tr>
 <td>Horizontal, submersible gel electrophoresis chamber with gel mold</td>
 <td>&nbsp;</td>
 <td>BioRad</td>
 <td>&nbsp;</td>
 <td>(or any comparable source)</td>
 </tr>
 <tr>
 <td>Power supply</td>
 <td>&nbsp;</td>
 <td>BioRad</td>
 <td>&nbsp;</td>
 <td>(or any comparable source)</td>
 </tr>
 <tr>
 <td>Molecular Imager Gel Doc System</td>
 <td>&nbsp;</td>
 <td>BioRad</td>
 <td>&nbsp;</td>
 <td>(or any comparable source)</td>
 </tr>
 </tbody>
</table>
Table 4. Materials

an allelotyping pcr for identifying salmonella enterica serovars enteritidis, hadar, heidelberg, and typhimurium

Current commercial PCRs tests for identifying Salmonella target genes unique to this genus. However, there are two species, six subspecies, and over 2,500 different Salmonella serovars, and not all are equal in their significance to public health. For example, finding S. enterica subspecies IIIa Arizona on a table egg layer farm is insignificant compared to the isolation of S. enterica subspecies I serovar Enteritidis, the leading cause of salmonellosis linked to the consumption of table eggs. Serovars are identified based on antigenic differences in lipopolysaccharide (LPS)(O antigen) and flagellin (H1 and H2 antigens). These antigenic differences are the outward appearance of the diversity of genes and gene alleles associated with this phenotype.
We have developed an allelotyping, multiplex PCR that keys on genetic differences between four major S. enterica subspecies I serovars found in poultry and associated with significant human disease in the US. The PCR primer pairs were targeted to key genes or sequences unique to a specific Salmonella serovar and designed to produce an amplicon with size specific for that gene or allele. Salmonella serovar is assigned to an isolate based on the combination of PCR test results for specific LPS and flagellin gene alleles. The multiplex PCRs described in this article are specific for the detection of S. enterica subspecies I serovars Enteritidis, Hadar, Heidelberg, and Typhimurium.
Here we demonstrate how to use the multiplex PCRs to identify serovar for a Salmonella isolate.

Watch this Scientific Journal Video about An Allelotyping PCR for Identifying Salmonella enterica serovars Enteritidis, Hadar, Heidelberg, and Typhimurium at JoVE.com

An Allelotyping PCR for Identifying Salmonella enterica serovars Enteritidis, Hadar, Heidelberg, and Typhimurium

We describe a multiplex PCR for the rapid detection of Salmonella enterica serovars Enteritidis, Hadar, Heidelberg, and Typhimurium. Specific Salmonella serovars can be identified by targeting a multiplex PCR to genes and sequences unique to the O-antigen biosynthesis cluster and flagellin of a given serovar. Serovar is assigned then to a Salmonella isolate based on the appearance of specific, size amplicons (PCR product) corresponding to the target allele.

An Allelotyping PCR for Identifying Salmonella enterica serovars Enteritidis, Hadar, Heidelberg, and Typhimurium

Current commercial PCRs tests for identifying Salmonella target genes unique to this genus.  However, there are two species, six subspecies, and over ...

an-allelotyping-pcr-for-identifying-salmonella-enterica-serovars

Research

JoVE Journal

Immunology and Infection

Identifying Dysregulated Genes Induced by Kaposi's Sarcoma-associated Herpesvirus (KSHV)

Currently KS is the most predominant HIV/AIDS related malignancy in Southern Africa and hence the world.1,2 It is characterized as an angioproliferative tumor of vascular endothelial cells and produces rare B cell lymphoproliferative diseases in the form of pleural effusion lymphomas (PEL) and some forms of multicentric Castleman's disease.3-5 Only 1-5% of cells in KS lesions actively support lytic replication of Kaposi's sarcoma-associated herpesvirus (KSHV), the etiological agent associated with KS, and it is clear that cellular factors must interact with viral factors in the process of oncogenesis and tumor progression.6,7 Identifying novel host-factor determinants which contribute to KS pathology is essential for developing prognostic markers for tumor progression and metastasis as well as for developing novel therapeutics for the treatment of KS.8 The accompanying video details the methods we use to identify host cell gene expression programs altered in dermal microvascular endothelial cells (DMVEC) after KSHV infection and in KS tumor tissue.9 Once dysregulated genes are identified by microarray analysis, changes in protein expression are confirmed by immunoblot and dual labeled immunofluorescence. Changes in transcriptional expression of dysregulated genes are confirmed in vitro by quantitative real-time polymerase chain reaction (qRT-PCR). Validation of in vitro findings using archival KS tumor tissue is also performed by dual labeled immunochemistry and tissue microarrays.8,10 Our approach to identifying dysregulated genes in the KS tumor tissue microenvironment will allow the development of in vitro and subsequently in vivo model systems for discovery and evaluation of potential novel therapeutic for the treatment of KS.

Identifying Dysregulated Genes Induced by Kaposi&#039;s Sarcoma-associated Herpesvirus KSHV

Host cell factors play a critical role in the establishment and maintenance of Kaposi's sarcoma (KS). We outline methods to identify host cell factors altered in KSHV-infected DMVEC cells, and in KS tumor tissue. Cellular genes altered by virus will serve as potential target(s) for novel therapeutics.

Currently KS is the most predominant HIV/AIDS related malignancy in Southern Africa and hence the world.1,2 It is characterized as an angioproliferative ...

An In vitro Model to Study Heterogeneity of Human Macrophage Differentiation and Polarization

Monocyte-derived macrophages represent an important cell type of the innate immune system. Mouse models studying macrophage biology suffer from the phenotypic and functional differences between murine and human monocyte-derived macrophages. Therefore, we here describe an in vitro model to generate and study primary human macrophages. Briefly, after density gradient centrifugation of peripheral blood drawn from a forearm vein, monocytes are isolated from peripheral blood mononuclear cells using negative magnetic bead isolation. These monocytes are then cultured for six days under specific conditions to induce different types of macrophage differentiation or polarization. The model is easy to use and circumvents the problems caused by species-specific differences between mouse and man. Furthermore, it is closer to the in vivo conditions than the use of immortalized cell lines. In conclusion, the model described here is suitable to study macrophage biology, identify disease mechanisms and novel therapeutic targets. Even though not fully replacing experiments with animals or human tissues obtained post mortem, the model described here allows identification and validation of disease mechanisms and therapeutic targets that may be highly relevant to various human diseases.

An In vitro Model to Study Heterogeneity of Human Macrophage Differentiation and Polarization

Monocyte-derived macrophages are important cells of the innate immune system. Here, we describe an easy to use in vitro model to generate these cells. Using gradient centrifugation, negative bead isolation and specific cell culture conditions, monocyte-derived macrophages can be generated for phenotypic and functional studies.

Monocyte-derived macrophages represent an important cell type of the innate immune system. Mouse models studying macrophage biology suffer from the ...

Measuring Growth and Gene Expression Dynamics of Tumor-Targeted S. Typhimurium Bacteria

The goal of these experiments is to generate quantitative time-course data on the growth and gene expression dynamics of attenuated S. typhimurium bacterial colonies growing inside tumors. 
We generated model xenograft tumors in mice by subcutaneous injection of a human ovarian cancer cell line, OVCAR-8 (NCI DCTD Tumor Repository, Frederick, MD). 
We transformed attenuated strains of S. typhimurium bacteria (ELH430:SL1344 phoPQ- 1) with a constitutively expressed luciferase (luxCDABE) plasmid for visualization2. These strains specifically colonize tumors while remaining essentially non-virulent to the mouse1. 
Once measurable tumors were established, bacteria were injected intravenously via the tail vein with varying dosage. Tumor-localized, bacterial gene expression was monitored in real time over the course of 60 hours using an in vivo imaging system (IVIS). At each time point, tumors were excised, homogenized, and plated to quantitate bacterial colonies for correlation with gene expression data. 
Together, this data yields a quantitative measure of the in vivo growth and gene expression dynamics of bacteria growing inside tumors.

Measuring Growth and Gene Expression Dynamics of Tumor-Targeted S. Typhimurium Bacteria

The goal of these experiments is to generate quantitative time-course data on the growth and gene expression dynamics of attenuated S. typhimurium bacterial colonies growing inside tumors. This video covers tumor cell preparation and implantation, bacteria preparation and injection, whole-animal luminescence imaging, tumor excision, and bacterial colony counting.

The goal of these experiments is to generate quantitative time-course data on the growth and gene expression dynamics of attenuated S. typhimurium ...

Procedures for Identifying Infectious Prions After Passage Through the Digestive System of an Avian Species

Infectious prion (PrPRes) material is likely the cause of fatal, neurodegenerative transmissible spongiform encephalopathy (TSE) diseases1. Transmission of TSE diseases, such as chronic wasting disease (CWD), is presumed to be from animal to animal2,3 as well as from environmental sources4-6. Scavengers and carnivores have potential to translocate PrPRes material through consumption and excretion of CWD-contaminated carrion. Recent work has documented passage of PrPRes material through the digestive system of American crows (Corvus brachyrhynchos), a common North American scavenger7.
We describe procedures used to document passage of PrPRes material through American crows. Crows were gavaged with RML-strain mouse-adapted scrapie and their feces were collected 4 hr post gavage. Crow feces were then pooled and injected intraperitoneally into C57BL/6 mice. Mice were monitored daily until they expressed clinical signs of mouse scrapie and were thereafter euthanized. Asymptomatic mice were monitored until 365 days post inoculation. Western blot analysis was conducted to confirm disease status. Results revealed that prions remain infectious after traveling through the digestive system of crows and are present in the feces, causing disease in test mice.

Scavengers have potential to translocate infectious transmissible spongiform encephalopathy prions in their feces to disease-free areas. We detail methods used to determine if mouse-adapted scrapie prions remain infectious after passage though the digestive tract of American crows (Corvus brachyrhynchos), a common consumer of dead animals.

Infectious prion (PrPRes) material is likely the cause of fatal, neurodegenerative transmissible spongiform encephalopathy (TSE) diseases1. Transmission ...

A Protocol to Infect Caenorhabditis elegans with Salmonella typhimurium

In the last decade, C. elegans has emerged as an invertebrate organism to study interactions between hosts and pathogens, including the host defense against gram-negative bacterium Salmonella typhimurium. Salmonella establishes persistent infection in the intestine of C. elegans and results in early death of infected animals. A number of immunity mechanisms have been identified in C. elegans to defend against Salmonella infections. Autophagy, an evolutionarily conserved lysosomal degradation pathway, has been shown to limit the Salmonella replication in C. elegans and in mammals. Here, a protocol is described to infect C. elegans with Salmonella typhimurium, in which the worms are exposed to Salmonella for a limited time, similar to Salmonella infection in humans. Salmonella infection significantly shortens the lifespan of C. elegans. Using the essential autophagy gene bec-1 as an example, we combined this infection method with C. elegans RNAi feeding approach and showed this protocol can be used to examine the function of C. elegans host genes in defense against Salmonella infection. Since C. elegans whole genome RNAi libraries are available, this protocol makes it possible to comprehensively screen for C. elegans genes that protect against Salmonella and other intestinal pathogens using genome-wide RNAi libraries.

A Protocol to Infect Caenorhabditis elegans with Salmonella typhimurium

C. elegans has emerged as a new genetic model to study host-pathogen interactions. Here we describe a protocol to infect C. elegans with Salmonella typhimurium coupled with the double-strand RNAi interference technique to examine the role of host genes in defense against Salmonella infection.

In the last decade, C. elegans has emerged as an invertebrate organism to study interactions between hosts and pathogens, including the host defense ...

High-throughput Assay to Phenotype Salmonella enterica Typhimurium Association, Invasion, and Replication in Macrophages

Salmonella species are zoonotic pathogens and leading causes of food borne illnesses in humans and livestock1. Understanding the mechanisms underlying Salmonella-host interactions are&#160;important to elucidate the molecular pathogenesis of Salmonella infection. The Gentamicin protection assay to phenotype Salmonella association, invasion and replication in phagocytic cells was adapted to allow high-throughput screening to define the roles of deletion mutants of Salmonella enterica serotype Typhimurium in host interactions using RAW 264.7 murine macrophages. Under this protocol, the variance in measurements is significantly reduced compared to the standard protocol, because wild-type and multiple mutant strains can be tested in the same culture dish and at the same time. The use of multichannel pipettes increases the throughput and enhances precision. Furthermore, concerns related to using less host cells per well in 96-well culture dish were addressed. Here, the protocol of the modified in vitro Salmonella invasion assay using phagocytic cells was successfully employed to phenotype 38 individual Salmonella deletion mutants for association, invasion and intracellular replication. The in vitro phenotypes are presented, some of which were subsequently confirmed to have in vivo phenotypes in an animal model. Thus, the modified, standardized assay to phenotype Salmonella association, invasion and replication in macrophages with high-throughput capacity could be utilized more broadly to study bacterial-host interactions.

High-throughput Assay to Phenotype Salmonella enterica Typhimurium Association, Invasion, and Replication in Macrophages

A high-throughput assay to in vitro phenotype Salmonella or other bacterial association, invasion, and replication in phagocytic cells with high-throughput capacity was developed. The method was employed to evaluate Salmonella gene knockout mutant strains for their involvements in host-pathogen interactions.

Salmonella species are zoonotic pathogens and leading causes of food borne illnesses in humans and livestock1. Understanding the mechanisms underlying ...

Establishment of a High-throughput Setup for Screening Small Molecules That Modulate c-di-GMP Signaling in Pseudomonas aeruginosa

Bacterial resistance to traditional antibiotics has driven research attempts to identify new drug targets in recently discovered regulatory pathways. Regulatory systems that utilize intracellular cyclic di-GMP (c-di-GMP) as a second messenger are one such class of target. c-di-GMP is a signaling molecule found in almost all bacteria that acts to regulate an extensive range of processes including antibiotic resistance, biofilm formation and virulence. The understanding of how c-di-GMP signaling controls aspects of antibiotic resistant biofilm development has suggested approaches whereby alteration of the cellular concentrations of the nucleotide or disruption of these signaling pathways may lead to reduced biofilm formation or increased susceptibility of the biofilms to antibiotics. We describe a simple high-throughput bioreporter protocol, based on green fluorescent protein (GFP), whose expression is under the control of the c-di-GMP responsive promoter cdrA, to rapidly screen for small molecules with the potential to modulate c-di-GMP cellular levels in Pseudomonas aeruginosa (P. aeruginosa). This simple protocol can screen upwards of 3,500 compounds within 48 hours and has the ability to be adapted to multiple microorganisms.

Establishment of a High-throughput Setup for Screening Small Molecules That Modulate c-di-GMP Signaling in Pseudomonas aeruginosa

This article describes the high-throughput assay that has been successfully established to screen large libraries of small molecules for their potential ability to manipulate cellular levels of cyclic di-GMP in Pseudomonas aeruginosa, providing a new powerful tool for antibacterial drug discovery and compound testing.

Bacterial resistance to traditional antibiotics has driven research attempts to identify new drug targets in recently discovered regulatory pathways. ...

Genotyping of Staphylococcus aureus by Ribosomal Spacer PCR (RS-PCR)

The ribosomal spacer PCR (RS-PCR) is a highly resolving and robust genotyping method for S. aureus that allows a high throughput at moderate costs and is, therefore, suitable to be used for routine purposes. For best resolution, data evaluation and data management, a miniaturized electrophoresis system is required. Together with such an electrophoresis system and the in-house developed software (freely available here) assignment of the pattern of bands to a genotype is standardized and straight forward. DNA extraction is simple (boiling prep), setting-up of the reactions is easy and they can be run on any standard PCR machine. PCR cycling is common except prolonged ramping and elongation times. Compared to spa typing and Multi Locus Sequence Typing (MLST), RS-PCR does not require DNA sequencing what simplifies the analysis considerably and allows a high throughput. Furthermore, the resolution for bovine strains of S. aureus is at least as good as spa typing and better than MLST or pulsed-field gel electrophoresis (PFGE). The RS-PCR data base includes presently a total of 141 genotypes and variants. The method is highly associated with the virulence gene pattern, contagiosity and pathogenicity of S. aureus strains involved in bovine mastitis. S. aureus genotype B (GTB) is contagious and causes herds problems causing large costs in the Switzerland and other European countries. All the other genotypes observed in Switzerland infect individual cows and quarters. Genotyping by RS-PCR allows the reliable prediction of the epidemiological and the pathogenic potential of S. aureus involved in bovine intramammary infection (IMI), two key factors for clinical veterinary medicine. Because of these beneficial properties together with moderate costs and a high sample throughput the goal of this publication is to give a detailed, step-by-step protocol for easily establishing and running RS-PCR for genotyping S. aureus in other laboratories.

Genotyping of Staphylococcus aureus by Ribosomal Spacer PCR RS-PCR

Here, ribosomal spacer PCR (RS-PCR) is used together with a miniaturized electrophoresis system as a fast and high resolution method for genotyping S. aureus at moderate costs allowing a high throughput.

The ribosomal spacer PCR (RS-PCR) is a highly resolving and robust genotyping method for S. aureus that allows a high throughput at moderate costs and is, ...

Isolation of Salmonella typhimurium-containing Phagosomes from Macrophages

Salmonella typhimurium is a facultative intracellular bacterium that causes gastroenteritis in humans. After invasion of the lamina propria, S. typhimurium bacteria are quickly detected and phagocytized by macrophages, and contained in vesicles known as phagosomes in order to be degraded. Isolation of S. typhimurium-containing phagosomes have been widely used to study how S. typhimurium infection alters the process of phagosome maturation to prevent bacterial degradation. Classically, the isolation of bacteria-containing phagosomes has been performed by sucrose gradient centrifugation. However, this process is time-consuming, and requires specialized equipment and a certain degree of dexterity. Described here is a simple and quick method for the isolation of S. typhimurium-containing phagosomes from macrophages by coating the bacteria with biotin-streptavidin-conjugated magnetic beads. Phagosomes obtained by this method can be suspended in any buffer of choice, allowing the utilization of isolated phagosomes for a broad range of assays, such as protein, metabolite, and lipid analysis. In summary, this method for the isolation of S. typhimurium-containing phagosomes is specific, efficient, rapid, requires minimum equipment, and is more versatile than the classical method of isolation by sucrose gradient-ultracentrifugation.

Isolation of Salmonella typhimurium-containing Phagosomes from Macrophages

We describe here a simple and quick method for the isolation of Salmonella typhimurium-containing phagosomes from macrophages by coating the bacteria with biotin and streptavidin.

Salmonella typhimurium is a facultative intracellular bacterium that causes gastroenteritis in humans. After invasion of the lamina propria, S. ...

A High-throughput Platform for the Screening of Salmonella spp./Shigella spp.

Fecal-oral transmission of acute gastroenteritis occurs from time to time, especially when people who handled food and water are infected by Salmonella spp./Shigella spp. The gold standard method for the detection of Salmonella spp./Shigella spp. is direct culture but this is labor-intensive and time-consuming. Here, we describe a high-throughput platform for Salmonella spp./Shigella spp. screening, using real-time polymerase chain reaction (PCR) combined with guided culture. There are two major stages: real-time PCR and the guided culture. For the first stage (real-time PCR), we explain each step of the method: sample collection, pre-enrichment, DNA extraction and real-time PCR. If the real-time PCR result is positive, then the second stage (guided culture) is performed: selective culture, biochemical identification and serological characterization. We also illustrate representative results generated from it. The protocol described here would be a valuable platform for the rapid, specific, sensitive and high-throughput screening of Salmonella spp./Shigella spp.

A High-throughput Platform for the Screening of Salmonella spp./Shigella spp.

Salmonella spp./Shigella spp. are common pathogens attributed to diarrhea. Here, we describe a high-throughput platform for the screening of Salmonella spp./Shigella spp. using real-time PCR combined with guided culture.

Fecal-oral transmission of acute gastroenteritis occurs from time to time, especially when people who handled food and water are infected by Salmonella ...