We would like to thank Brandon Hassett and Carlos Ortiz for their technical assistance. This work was supported by the NSF grants IOB-0544428, IOS-0951272, and IOS-0925561 to Dr. Michael Kolomiets, and by the USDA National Institute of Food and Agriculture (NIFA), AFRI Plant Breeding and Education Grant #2010-85117-20539 to Drs. Seth Murray, Thomas Isakeit, and Michael Kolomiets.

1. Maize Kernel Bioassay
<ol>
	<li>Two weeks prior, culture the fungal pathogens on Potato Dextrose Agar (PDA) at 28 &deg;C.</li>
	<li>Select kernels with similar size and shape, preferably flattened so they lay level with the bottom of bioassay vials, and place in 50 ml falcon tubes. Kernels selected must have been produced concurrently in same environment to ensure similar seed age and metabolite composition.</li>
	<li>Surface sterilize kernels by shaking tubes at room temperature for 5 minutes with 70% ethanol, 1 minute with sterile water, and 10 minutes with 6% sodium hypochlorite. Rinse, three times, for 5 minutes each time, with sterile water while continuing shaking. Pat kernels dry on autoclaved towels.</li>
	<li>To facilitate infection of Aspergillus flavus, create a small wound on the embryo-side with an 18 G needle to the depth of 0.5 cm. However, in our experience a larger wound is needed for Fusarium verticillioides infection. Therefore, use a razor blade to cut into the embryo-side at a depth of 0.5 mm.</li>
	<li>Prepare inoculum suspension by scraping cultured plates in about 5 ml of autoclaved 0.01% Tween-20 solution to liberate spores and remove mycelium by gravity filtering through at least 4 layers of autoclaved cheesecloth. Calculate spore concentration with hemocytometer and adjust to 106 spores /ml for A. flavus or F. verticillioides. If using other species of pathogen or host, it is important to evaluate concentrations of inoculum for appropriate infection.</li>
	<li>Create a humidity chamber in a plastic container (29.2 cm X 18.7 cm X 8.3 cm) lined with five sheets of paper towels and 100 ml of sterile water. Chamber is not water- or air-tight and additional water should be added throughout experiment to keep paper towels moist.</li>
	<li>Place four kernels into an autoclaved 20 ml glass scintillation vial and record their mass. Inoculate with 200 &mu;l spore suspension, cap, and vortex to evenly coat kernels with suspension. Loosen caps to freely allow air exchange and place into humidity chamber.</li>
</ol>
Note: For seeds or fungi other than those described in these methods, the amount of inoculum required may be different and should be derived experimentally.
<ol start="8">
	<li>Incubate kernels under a 12-h-light/12-h-dark photoperiod at 28 &deg;C for 7 days or until desired.</li>
</ol>
2. Conidia Enumeration
<ol>
	<li>After the infection period, add 5 ml of autoclaved 0.01% Tween-20 to scintillation vials containing infected kernels and vortex thoroughly for 1 minute.</li>
	<li>Using a wide-bore pipette tip, immediately dilute by transferring two separate 200 &mu;l aliquots of spore suspension into 2.0 ml Eppendorf tubes containing 1.8 ml of 0.01% Tween-20 (or dilute as needed depending on infection).</li>
	<li>Enumerate each 200 &mu;l aliquot twice with a hemocytometer4 and compare levels of conidia between treatments.</li>
</ol>
3. Aflatoxin Quantification
<ol>
	<li>Add kernels from 1 sample to 50 ml blender cup with 20 ml of 80% methanol and 0.05 g NaCl, cover and blend at high speed for 1 min.</li>
	<li>Place a fluted filter paper over a clean collection vessel and pour extract into filter.</li>
	<li>Transfer 10 ml of filtrate to a clean vessel, add 20 ml distilled H2O, and mix well.</li>
	<li>Filter sample through a 1.5 &mu;m glass microfibre filter into a clean cup.</li>
	<li>Apply 1 ml of filtered extract to Afla Test column and, using compressed air, force the filtered extract through the column at a rate of 1-2 drops/second until emptied.</li>
	<li>Wash column twice with 1 ml of distilled H2O and pass through column at 1-2 drop/second until air comes through.</li>
	<li>Elute column with 1ml of 100% HPLC grade methanol at a 1-2 drop/second rate collected into a glass cuvette.</li>
	<li>Add 1 ml of Afla Test Developer to eluate and mix well. Place cuvette into calibrated fluorometer and read at 60 sec.</li>
</ol>
Note: When using the Aflatest FGIS protocol, the measurement from the fluorometer is calculated based upon an initial 50 grams of sample extracted in 100 ml. If you consider the dilution of the sample with water, the 1 ml applied to a column represents 0.166 grams of sample. So, when modifying the protocol, you need to take into account the differences in sample size and the initial extraction solution. For example, 2 grams of kernels are extracted in 20 ml 80% methanol. This is 0.1 grams/ml. When 1 ml of this sample is mixed with 2 ml water, the proportion of sample in the liquid is now 0.033 grams/ml. If this sample gives a fluorometer reading of 100 ppb, the actual concentration is based on the proportion of sample to 0.166, that is, ppb = (0.166 gram X 100 ppb)/ 0.1 gram), or 166 ppb. For alternative aflatoxin quantification methods, see reference 7.
4. Fumonisin B1 (FB1) Analysis
<ol>
	<li>When fungus is grown on corn kernels, extract samples overnight with 10 ml of acetonitrile/water (50/50, v/v) at room temperature without agitation. Subsequently, mix the extract (2 ml) with deionized water (6 ml), and apply this mix (8 ml) directly to the C-18 solid phase extraction.</li>
	<li>Before loading sample, precondition the C-18 solid phase extraction column by rinsing with 2 ml of acetonitrile followed by 2 ml of water.</li>
	<li>After loading the sample, the column is washed with 2 ml of water followed by 2 ml of acetonitrile/water (15/85, v/v). Sample for HPLC analysis (containing FB1) is eluted with 2 ml of acetonitrile/water (70/30, v/v).</li>
	<li>Derivatize FB1 with o-phthaldehyde (OPA) by transferring 0.1 ml of the column eluate to a vial containing 0.1 ml borate buffer (0.05 M boric acid/0.05 M sodium borate [50/50, v/v], pH 8.5) and 0.1 ml OPA (0.1 mg/ml in acetonitrile with 0.5% - mercaptoethanol).</li>
	<li>Stop the reaction after 10 min by adding 0.5 ml of acetonitrile/0.01 M boric acid (40/60, v/v).</li>
	<li>Analyze FB1 on a Shimadzu HPLC LC-20AT system equipped with an analytical Zorbax ODS column (4.6 150 mm) and a variable wavelength Shimatzu RF-10Axl fluorescence detector (excitation 335 nm/emission 440 nm).</li>
	<li>A linear gradient is used (solvent A: acetonitrile/0.1 M sodium phosphate (40/60), pH 3.3; solvent B: acetonitrile/ 0.1 M sodium phosphate (60/40), pH 3.3) and the gradient program is as follows: 100% A to 100% B in 10 min, 100% B for 5 min.</li>
	<li>Analyze FB1 standards by HPLC, and peak area measurements are used to generate standard curve. Subsequently, quantify FB1 level in a sample by comparing peak areas with FB1 standard curve.</li>
</ol>
5. Ergosterol Analysis
<ol>
	<li>Culture the fungus on corn kernels for 7-14 days.</li>
	<li>After the incubation period, add 10 ml of chloroform: methanol (2:1, v/v) in each vial. Once added, shake samples well and then incubate vials in the dark at room temperature for 24 hrs.</li>
	<li>After 24 hrs, centrifuge the samples, and collect the supernatant and filter it through 0.45 um nylon membrane.</li>
	<li>Inject the sample directly into a Shimadzu HPLC LC-20AT system equipped with a 4.6 U ODS-C18 column (200 &Aring;, 250 &plusmn; 4.6 mm) and a Shimadzu SPD-20A UV/VIS detector set to monitor at 282 nm.</li>
	<li>Use methanol (100%) at a flow rate of 1.5 mL/min as the mobile phase. Determine quantification of ergosterol in samples by comparing peak areas of samples to a standard curve generated from HPLC-grade ergosterol.</li>
</ol>
6. Representative Results
Following inoculation and incubation in a humidity chamber for two to three days, fungal growth should begin to appear on the kernels. Seven days post treatment, vegetative growth on treated plants should be clearly visible, while mock controls should be uninfected (Figure 1B, top). Periods of longer incubation are conducive to more copious vegetative growth (Figure 1B, bottom). Under the conditions described herein (Figure 2G), A. flavus wild-type NRRL 3357 displayed maximum values of colonization, aflatoxin accumulation, and conidia production at 8, 6, and 8 days, respectively (Figure 2, A-C). However, when mycotoxin contamination and conidiation were compared per unit of ergosterol, the greatest levels were observed at 4 and 6 days, respectively (Figure 2, D-E). Figure 2F summarizes these fungal biomass-dependent maximum-value observations. Interestingly, between 4-6 days post-inoculation, the kernels undergo nucleic acid degradation, as seen by total RNA from samples on the time-course (Figure 2H, bottom).
Several studies, including our own, have examined F. verticilliodies infection on maize kernels2,8,9,10,11,12 and successfully used 7-13 days as time-points for observations. Using the methods described herein, fumonisin levels range from 3,500-8,000 ng/g kernel4,13 and ergosterol levels range from 5,000-10,000 ng/g kernel14,13. Figure 3 shows representative peaks for fumonisin (top) and ergosterol (bottom) from HPLC chromatograms. Measurement of ergosterol can also be carried out via absorbance spectra 15 .
<img alt="Figure 1" src="/files/ftp_upload/3727/3727fig1.jpg" /> 
Figure 1. Flow chart for the quantification of fungal biomass, sporogenesis, and mycotoxin production and representative results for vegetative growth from kernel bioassays. A) Flow chart outlining the described method for quantification of fundamental biological parameters used to assess fungal pathogenesis on maize kernels. B) Representative results for kernel bioassays. Top, A. flavus (NRRL3357) vegetative growth on maize kernels in the B73 genetic background. Seeds were inoculated with 200 &mu;l of 106 spores/mL and pictures were taken 7 days post inoculation. Bottom, F. verticillioides inoculated kernels in B73 background 13 days post infection. Kernels were inoculated with 200 &mu;l of 106 spores/mL.
<img alt="Figure 2" src="/files/ftp_upload/3727/3727fig2.jpg" /> 
Figure 2. Aspergillus flavus kernel bioassay time-course. B73 kernels were inoculated with 200 &mu;l of 106 spores/ml Aspergillus flavus conidial suspension and incubated for 2-8 days (G). All values were determined from the dry weight average of 4 kernels (n = 3-4; mean &plusmn; SE). A) Colonization (based on ergosterol), (B) aflatoxin, and (C) conidiation were quantified using methods described above. E) Aflatoxin and (F) conidiation are displayed as a function of fungal biomass as measured through h ergosterol. F) Maximum values for ergosterol, aflatoxin accumulation, and conidiation as a function of fungal biomass - maximum values for each quantity observed was set to 100%. (H) 1 &mu;g per lane of total RNA from time-course.
<img alt="Figure 3" src="/files/ftp_upload/3727/3727fig3.jpg" /> 
Figure 3. Representative High Performance Liquid Chromatography (HPLC) chromatograms for fumonisin B1 (top) and ergosterol (bottom) isolated from kernels infected with Fusarium verticillioides (M3125).

<ol>
	<li>Tsitsigiannis, D. I., Keller, N. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Oxylipins+as+developmental+and+host-fungal+communication+signals.">Oxylipins as developmental and host-fungal communication signals.</a> Trends Microbiol. 15, 109-118 (2007).</li><li>Gao, X., Shim, W. -. B., G&#246;bel, C., Kunze, S., Feussner, I., Meeley, R., Balint-Kurti, P., Kolomiets, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Disruption+of+a+maize+9-lipoxygenase+results+in+increased+resistance+to+fungal+pathogens+and+reduced+levels+of+contamination+with+mycotoxin+fumonisin.">Disruption of a maize 9-lipoxygenase results in increased resistance to fungal pathogens and reduced levels of contamination with mycotoxin fumonisin.</a> Mol. Plant-Microbe Interact. 20, 922-933 (2007).</li><li>Brodhagen, M., Tsitsigiannis, D. I., Hornung, E., Goebel, C., Feussner, I., Keller, N. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Reciprocal+oxylipin-mediated+cross-talk+in+the+Aspergillus+-+seed+pathosystem.">Reciprocal oxylipin-mediated cross-talk in the Aspergillus - seed pathosystem.</a> Mol. Microbiol. 67, 378-391 (2008).</li><li>Gao, X., Brodhagen, M., Isakeit, T., Brown, S. H., G&#246;bel, C., Betran, J., Feussner, I., Keller, N. P., Kolomiets, M. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Inactivation+of+the+lipoxygenase+ZmLOX3+increases+susceptibility+of+maize+to+Aspergillus+spp.">Inactivation of the lipoxygenase ZmLOX3 increases susceptibility of maize to Aspergillus spp.</a> Mol. Plant-Microbe Interact. 22, 222-231 (2009).</li><li>Gao, X. Q., Kolomiets, M. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Host-derived+lipids+and+oxylipins+are+crucial+signals+in+modulating+mycotoxin+production+by+fungi.">Host-derived lipids and oxylipins are crucial signals in modulating mycotoxin production by fungi.</a> Toxin Rev. 28, 79-88 (2009).</li><li>Mukherjee, M., Kim, J. -. E., Park, Y. -. S., Kolomiets, M. V., Shim, W. -. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Regulators+of+G+protein+signaling+in+F.+verticillioides+mediate+differential+host-pathogen+responses+on+non-viable+versus+viable+maize+kernels.">Regulators of G protein signaling in F. verticillioides mediate differential host-pathogen responses on non-viable versus viable maize kernels.</a> Mol. 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Agric. and Food Chem. 5, 360-363 (1957).</li></ol>

I have nothing to disclose.

The methods described here have been tested extensively and proven to be robust in the generation of quantifiable results for fungal colonization, sporogenesis, and production of mycotoxins. Moreover, these methods should be applicable to seeds from other plant species that are susceptible to contamination with mycotoxigenic fungi (e.g. peanuts, wheat, cotton, pistachios, etc.). For competent plant-pathogen interaction analyses, it is imperative that the seeds be kept alive. A small wound site on the embryo side of the ...

<table border="1">
<tbody>
<tr>
 <td>Name of the reagent</td>
 <td>Company</td>
 <td>Catalog #</td>
</tr>
<tr>
 <td>Potato Dextrose Agar</td>
 <td>Fisher Scientifc</td>
 <td>S71659A</td>
</tr>
<tr>
 <td>Tween-20</td>
 <td>Fisher Scientifc</td>
 <td>BP337-100</td>
</tr>
<tr>
 <td>Plastic incubation container</td>
 <td>Sterilite</td>
 <td>1713LAB06</td>
</tr>
<tr>
 <td>Blender</td>
 <td>Vicam</td>
 <td>20200</td>
</tr>
<tr>
 <td>24 cm Fluted Filter Papers</td>
 <td>Vicam</td>
 <td>31240</td>
</tr>
<tr>
 <td>1.5 &mu;m glass microfibre</td>
 <td>Vicam</td>
 <td>31955</td>
</tr>
<tr>
 <td>Afla Test column</td>
 <td>Vicam</td>
 <td>G1024</td>
</tr>
<tr>
 <td>Afrla Test Developer</td>
 <td>Vicam</td>
 <td>32010</td>
</tr>
<tr>
 <td>Methanol</td>
 <td>Vicam</td>
 <td>35016</td>
</tr>
<tr>
 <td>Acetonitrile</td>
 <td>Fisher Scientifc</td>
 <td>AC14952-0025</td>
</tr>
<tr>
 <td>Ethanol</td>
 <td>Fisher Scientifc</td>
 <td>AC39769-0025</td>
</tr>
<tr>
 <td>C-18 solid phase extraction column (Prep SEP SPE C18 Column)</td>
 <td>Fisher Scientifc</td>
 <td>60108-304</td>
</tr>
<tr>
 <td>O-phthalaldehyde (OPA)</td>
 <td>Sigma Chemical Co</td>
 <td>79760-5g</td>
</tr>
<tr>
 <td>Boric acid</td>
 <td>Fisher Scientifc</td>
 <td>BP168-500</td>
</tr>
<tr>
 <td>Sodium borate</td>
 <td>Fisher Scientifc</td>
 <td>RDCS0330500</td>
</tr>
<tr>
 <td>Mercaptoethanol</td>
 <td>Fisher Scientifc</td>
 <td>45-000-231</td>
</tr>
<tr>
 <td>Shimadzu HPLC LC-20AT (Pump)</td>
 <td>Shimadzu Scientific Instruments, Inc.</td>
 <td>LC-20AT</td>
</tr>
<tr>
 <td>Zorbax ODS column (4.6x150mm)</td>
 <td>Agilent Technologies</td>
 <td>443905-902</td>
</tr>
<tr>
 <td>Shimatzu RF-10Axl fluorescence detector</td>
 <td>Shimadzu Scientific Instruments, Inc.</td>
 <td>RF-10AXL</td>
</tr>
<tr>
 <td>Sodium phosphate</td>
 <td>Fisher Scientifc</td>
 <td>AC38987-0010</td>
</tr>
<tr>
 <td>FB1 standards</td>
 <td>Sigma Chemical Co.</td>
 <td>F1147-1mg</td>
</tr>
<tr>
 <td>Chloroform</td>
 <td>VWR</td>
 <td>MK444410</td>
</tr>
<tr>
 <td>13 mm syringe filter with 0.45 um nylon membrane (HPLC)</td>
 <td>Pall Life Science</td>
 <td>4426</td>
</tr>
<tr>
 <td>Ergosterol</td>
 <td>Sigma-Aldrich</td>
 <td>45480-50G-F</td>
</tr>
<tr>
 <td>Scintillation vials</td>
 <td>VWR</td>
 <td>66021-602</td>
</tr>
<tr>
 <td>Sodium Chloride</td>
 <td>Vicam</td>
 <td>G1124</td>
</tr>
</tbody>
</table>

quantification of fungal colonization, sporogenesis, and production of mycotoxins using kernel bioassays

The rotting of grains by seed-infecting fungi poses one of the greatest economic challenges to cereal production worldwide, not to mention serious risks to human and animal health. Among cereal production, maize is arguably the most affected crop, due to pathogen-induced losses in grain integrity and mycotoxin seed contamination. The two most prevalent and problematic mycotoxins for maize growers and food and feed processors are aflatoxin and fumonisin, produced by Aspergillus flavus and Fusarium verticillioides, respectively.
Recent studies in molecular plant-pathogen interactions have demonstrated promise in understanding specific mechanisms associated with plant responses to fungal infection and mycotoxin contamination1,2,3,4,5,6. Because many labs are using kernel assays to study plant-pathogen interactions, there is a need for a standardized method for quantifying different biological parameters, so results from different laboratories can be cross-interpreted. For a robust and reproducible means for quantitative analyses on seeds, we have developed in-lab kernel assays and subsequent methods to quantify fungal growth, biomass, and mycotoxin contamination. Four sterilized maize kernels are inoculated in glass vials with a fungal suspension (106) and incubated for a predetermined period. Sample vials are then selected for enumeration of conidia by hemocytometer, ergosterol-based biomass analysis by high performance liquid chromatography (HPLC), aflatoxin quantification using an AflaTest fluorometer method, and fumonisin quantification by HPLC.

Watch this Scientific Journal Video about Quantification of Fungal Colonization, Sporogenesis, and Production of Mycotoxins Using Kernel Bioassays at JoVE.com

Quantification of Fungal Colonization, Sporogenesis, and Production of Mycotoxins Using Kernel Bioassays

The devastation of cereal crops by seed-infecting fungi has prompted numerous research efforts to better understand plant-pathogen interactions. To study seed-fungal interactions in a laboratory setting, we developed a robust method for the quantification of fungal reproduction, biomass, and mycotoxin contamination using kernel bioassays.

The rotting of grains by seed-infecting fungi poses one of the greatest economic challenges to cereal production worldwide, not to mention serious risks ...

quantification-fungal-colonization-sporogenesis-production-mycotoxins

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JoVE Journal

Immunology and Infection

18.0K Views.  Texas A&M University. La devastazione delle colture di cereali da semi-infezione funghi ha spinto numerosi sforzi di ricerca per comprendere meglio le interazioni pianta-patogeno. Per studiare le interazioni semi-fungine in un ambiente di laboratorio, abbiamo sviluppato un metodo affidabile per la quantificazione della riproduzione fungina, la biomassa, e la contaminazione da micotossine mediante biotest del kernel.

Video: Quantification of Fungal Colonization, Sporogenesis, and Production of Mycotoxins Using Kernel Bioassays

Colonization of Euprymna scolopes Squid by Vibrio fischeri

Specific bacteria are found in association with animal tissue1-5. Such host-bacterial associations (symbioses) can be detrimental (pathogenic), have no fitness consequence (commensal), or be beneficial (mutualistic). While much attention has been given to pathogenic interactions, little is known about the processes that dictate the reproducible acquisition of beneficial/commensal bacteria from the environment. The light-organ mutualism between the marine Gram-negative bacterium V. fischeri and the Hawaiian bobtail squid, E. scolopes, represents a highly specific interaction in which one host (E. scolopes) establishes a symbiotic relationship with only one bacterial species (V. fischeri) throughout the course of its lifetime6,7. Bioluminescence produced by V. fischeri during this interaction provides an anti-predatory benefit to E. scolopes during nocturnal activities8,9, while the nutrient-rich host tissue provides V. fischeri with a protected niche10. During each host generation, this relationship is recapitulated, thus representing a predictable process that can be assessed in detail at various stages of symbiotic development. In the laboratory, the juvenile squid hatch aposymbiotically (uncolonized), and, if collected within the first 30-60 minutes and transferred to symbiont-free water, cannot be colonized except by the experimental inoculum6. This interaction thus provides a useful model system in which to assess the individual steps that lead to specific acquisition of a symbiotic microbe from the environment11,12.
Here we describe a method to assess the degree of colonization that occurs when newly hatched aposymbiotic E. scolopes are exposed to (artificial) seawater containing V. fischeri. This simple assay describes inoculation, natural infection, and recovery of the bacterial symbiont from the nascent light organ of E. scolopes. Care is taken to provide a consistent environment for the animals during symbiotic development, especially with regard to water quality and light cues. Methods to characterize the symbiotic population described include (1) measurement of bacterially-derived bioluminescence, and (2) direct colony counting of recovered symbionts.

Colonization of Euprymna scolopes Squid by Vibrio fischeri

The method outlines the procedure by which the Hawaiian bobtail squid, Euprymna scolopes and its bacterial symbiont, Vibrio fischeri, are raised separately and then introduced to allow for specific colonization of the squid light organ by the bacteria. Colonization detection by bacterially-derived luminescence and by direct colony counting are described.

Specific bacteria are found in association with animal tissue1-5. Such host-bacterial associations (symbioses) can be detrimental (pathogenic), have no ...

Live-cell Video Microscopy of Fungal Pathogen Phagocytosis

Phagocytic clearance of fungal pathogens, and microorganisms more generally, may be considered to consist of four distinct stages: (i) migration of phagocytes to the site where pathogens are located; (ii) recognition of pathogen-associated molecular patterns (PAMPs) through pattern recognition receptors (PRRs); (iii) engulfment of microorganisms bound to the phagocyte cell membrane, and (iv) processing of engulfed cells within maturing phagosomes and digestion of the ingested particle. Studies that assess phagocytosis in its entirety are informative1, 2, 3, 4, 5 but are limited in that they do not normally break the process down into migration, engulfment and phagosome maturation, which may be affected differentially. Furthermore, such studies assess uptake as a single event, rather than as a continuous dynamic process. We have recently developed advanced live-cell imaging technologies, and have combined these with genetic functional analysis of both pathogen and host cells to create a cross-disciplinary platform for the analysis of innate immune cell function and fungal pathogenesis. These studies have revealed novel aspects of phagocytosis that could only be observed using systematic temporal analysis of the molecular and cellular interactions between human phagocytes and fungal pathogens and infectious microorganisms more generally. For example, we have begun to define the following: (a) the components of the cell surface required for each stage of the process of recognition, engulfment and killing of fungal cells1, 6, 7, 8; (b) how surface geometry influences the efficiency of macrophage uptake and killing of yeast and hyphal cells7; and (c) how engulfment leads to alteration of the cell cycle and behavior of macrophages 9, 10.
In contrast to single time point snapshots, live-cell video microscopy enables a wide variety of host cells and pathogens to be studied as continuous sequences over lengthy time periods, providing spatial and temporal information on a broad range of dynamic processes, including cell migration, replication and vesicular trafficking. Here we describe in detail how to prepare host and fungal cells, and to conduct the video microscopy experiments. These methods can provide a user-guide for future studies with other phagocytes and microorganisms.

We describe methods for live-cell video microscopy of Candida albicans phagocytosis by macrophages. These methods enable stage-specific analysis of macrophage migration, recognition, engulfment and phagosome maturation and reveal novel aspects of phagocytosis.

Phagocytic clearance of fungal pathogens, and microorganisms more generally, may be considered to consist of four distinct stages: (i) migration of ...

Characterization of Inflammatory Responses During Intranasal Colonization with Streptococcus pneumoniae

Nasopharyngeal colonization by Streptococcus pneumoniae is a prerequisite to invasion to the lungs or bloodstream1. This organism is capable of colonizing the mucosal surface of the nasopharynx, where it can reside, multiply and eventually overcome host defences to invade to other tissues of the host. Establishment of an infection in the normally lower respiratory tract results in pneumonia. Alternatively, the bacteria can disseminate into the bloodstream causing bacteraemia, which is associated with high mortality rates2, or else lead directly to the development of pneumococcal meningitis. Understanding the kinetics of, and immune responses to, nasopharyngeal colonization is an important aspect of S. pneumoniae infection models.
Our mouse model of intranasal colonization is adapted from human models3 and has been used by multiple research groups in the study of host-pathogen responses in the nasopharynx4-7. In the first part of the model, we use a clinical isolate of S. pneumoniae to establish a self-limiting bacterial colonization that is similar to carriage events in human adults. The procedure detailed herein involves preparation of a bacterial inoculum, followed by the establishment of a colonization event through delivery of the inoculum via an intranasal route of administration. Resident macrophages are the predominant cell type in the nasopharynx during the steady state. Typically, there are few lymphocytes present in uninfected mice8, however mucosal colonization will lead to low- to high-grade inflammation (depending on the virulence of the bacterial species and strain) that will result in an immune response and the subsequent recruitment of host immune cells. These cells can be isolated by a lavage of the tracheal contents through the nares, and correlated to the density of colonization bacteria to better understand the kinetics of the infection.

Characterization of Inflammatory Responses During Intranasal Colonization with Streptococcus pneumoniae

Colonization of the murine nasopharynx with Streptococcus pneumoniae and the subsequent extraction of adherent or recruited cells is described. This technique involves flushing the nasopharynx and collection of the fluid through the nares and is adaptable for various readouts, including differential cell quantification and analysis of mRNA expression in situ.

Nasopharyngeal colonization by Streptococcus pneumoniae is a prerequisite to invasion to the lungs or bloodstream1. This organism is capable of colonizing ...

Isolation, Purification and Labeling of Mouse Bone Marrow Neutrophils for Functional Studies and Adoptive Transfer Experiments

Neutrophils are critical effector cells of the innate immune system. They are rapidly recruited at sites of acute inflammation and exert protective or pathogenic effects depending on the inflammatory milieu. Nonetheless, despite the indispensable role of neutrophils in immunity, detailed understanding of the molecular factors that mediate neutrophils' effector and immunopathogenic effects in different infectious diseases and inflammatory conditions is still lacking, partly because of their short half life, the difficulties with handling of these cells and the lack of reliable experimental protocols for obtaining sufficient numbers of neutrophils for downstream functional studies and adoptive transfer experiments. Therefore, simple, fast, economical and reliable methods are highly desirable for harvesting sufficient numbers of mouse neutrophils for assessing functions such as phagocytosis, killing, cytokine production, degranulation and trafficking. To that end, we present a reproducible density gradient centrifugation-based protocol, which can be adapted in any laboratory to isolate large numbers of neutrophils from the bone marrow of mice with high purity and viability. Moreover, we present a simple protocol that uses CellTracker dyes to label the isolated neutrophils, which can then be adoptively transferred into recipient mice and tracked in several tissues for at least 4 hr post-transfer using flow cytometry. Using this approach, differential labeling of neutrophils from wild-type and gene-deficient mice with different CellTracker dyes can be successfully employed to perform competitive repopulation studies for evaluating the direct role of specific genes in trafficking of neutrophils from the blood into target tissues in vivo.

We describe a protocol for isolation and purification of neutrophils from mouse bone marrow by density gradient centrifugation and for neutrophil labeling using CellTracker dyes. This represents a simple, fast, reproducible and economical method for obtaining large numbers of neutrophils for downstream functional studies or adoptive transfer and tracking experiments.

Neutrophils are critical effector cells of the innate immune system. They are rapidly recruited at sites of acute inflammation and exert protective or ...

Establishment of an In vitro System to Study Intracellular Behavior of Candida glabrata in Human THP-1 Macrophages

A cell culture model system, if a close mimic of host environmental conditions, can serve as an inexpensive, reproducible and easily manipulatable alternative to animal model systems for the study of a specific step of microbial pathogen infection. A human monocytic cell line THP-1 which, upon phorbol ester treatment, is differentiated into macrophages, has previously been used to study virulence strategies of many intracellular pathogens including Mycobacterium tuberculosis. Here, we discuss a protocol to enact an in vitro cell culture model system using THP-1 macrophages to delineate the interaction of an opportunistic human yeast pathogen Candida glabrata with host phagocytic cells. This model system is simple, fast, amenable to high-throughput mutant screens, and requires no sophisticated equipment. A typical THP-1 macrophage infection experiment takes approximately 24 hr with an additional 24-48 hr to allow recovered intracellular yeast to grow on rich medium for colony forming unit-based viability analysis. Like other in vitro model systems, a possible limitation of this approach is difficulty in extrapolating the results obtained to a highly complex immune cell circuitry existing in the human host. However, despite this, the current protocol is very useful to elucidate the strategies that a fungal pathogen may employ to evade/counteract antimicrobial response and survive, adapt, and proliferate in the nutrient-poor environment of host immune cells.

Establishment of an In vitro System to Study Intracellular Behavior of Candida glabrata in Human THP-1 Macrophages

The current article outlines a protocol to establish an in vitro cell culture model system to study the interaction of a facultative intracellular human fungal pathogen Candida glabrata with human macrophages which will be a useful tool to advance our knowledge of fungal virulence mechanisms.

A cell culture model system, if a close mimic of host environmental conditions, can serve as an inexpensive, reproducible and easily manipulatable ...

Investigating the Effects of Probiotics on Pneumococcal Colonization Using an In Vitro Adherence Assay

Adherence of Streptococcus pneumoniae (the pneumococcus) to the epithelial lining of the nasopharynx can result in colonization and is considered a prerequisite for pneumococcal infections such as pneumonia and otitis media. In vitro adherence assays can be used to study the attachment of pneumococci to epithelial cell monolayers and to investigate potential interventions, such as the use of probiotics, to inhibit pneumococcal&#160;colonization.&#160;The protocol described here is used to investigate the effects of the probiotic Streptococcus salivarius on the adherence of pneumococci to the human epithelial cell line CCL-23 (sometimes referred to as HEp-2 cells). The assay involves three main steps: 1) preparation of epithelial and bacterial cells, 2) addition of bacteria to epithelial cell monolayers, and 3) detection of adherent pneumococci by viable counts (serial dilution and plating) or quantitative real-time PCR (qPCR). This technique is relatively straightforward and does not require specialized equipment other than a tissue culture setup. The assay can be used to test other probiotic species and/or potential inhibitors of pneumococcal colonization and can be easily modified to address other scientific questions regarding pneumococcal adherence and invasion.

Investigating the Effects of Probiotics on Pneumococcal Colonization Using an In Vitro Adherence Assay

In vitro adherence assays can be used to study the attachment of Streptococcus pneumoniae to epithelial cell monolayers and to investigate potential interventions such as the use of probiotics for inhibiting pneumococcal colonization.

Adherence of Streptococcus pneumoniae (the pneumococcus) to the epithelial lining of the nasopharynx can result in colonization and is considered a ...

Assessing Bacterial Invasion of Cardiac Cells in Culture and Heart Colonization in Infected Mice Using Listeria monocytogenes

Listeria monocytogenes is a Gram-positive facultative intracellular pathogen that is capable of causing serious invasive infections in immunocompromised patients, the elderly, and pregnant women. The most common manifestations of listeriosis in humans include meningitis, encephalitis, and fetal abortion. A significant but much less documented sequelae of invasive L. monocytogenes infection involves the heart. The death rate from cardiac illness can be up to 35% despite treatment, however very little is known regarding L. monocytogenes colonization of cardiac tissue and its resultant pathologies. In addition, it has recently become apparent that subpopulations of L. monocytogenes have an enhanced capacity to invade and grow within cardiac tissue. This protocol describes in detail in vitro and in vivo methods that can be used for assessing cardiotropism of L. monocytogenes isolates. Methods are presented for the infection of H9c2 rat cardiac myoblasts in tissue culture as well as for the determination of bacterial colonization of the hearts of infected mice. These methods are useful not only for identifying strains with the potential to colonize cardiac tissue in infected animals, but may also facilitate the identification of bacterial gene products that serve to enhance cardiac cell invasion and/or drive changes in heart pathology. These methods also provide for the direct comparison of cardiotropism between multiple L. monocytogenes strains.

Assessing Bacterial Invasion of Cardiac Cells in Culture and Heart Colonization in Infected Mice Using Listeria monocytogenes

Listeria monocytogenes causes fetal infections in pregnant women and meningitis in susceptible populations. Subpopulations of bacteria can colonize cardiac tissue, causing myocarditis in patients and laboratory animals. Here we present a protocol that describes how to assess L. monocytogenes cardiac cell invasion in vitro and cardiac colonization in infected animals.

Listeria monocytogenes is a Gram-positive facultative intracellular pathogen that is capable of causing serious invasive infections in immunocompromised ...

A Murine Model of Group B Streptococcus Vaginal Colonization

Streptococcus agalactiae (group B Streptococcus, GBS), is a Gram-positive, asymptomatic colonizer of the human gastrointestinal tract and vaginal tract of 10 - 30% of adults. In immune-compromised individuals, including neonates, pregnant women, and the elderly, GBS may switch to an invasive pathogen causing sepsis, arthritis, pneumonia, and meningitis. Because GBS is a leading bacterial pathogen of neonates, current prophylaxis is comprised of late gestation screening for GBS vaginal colonization and subsequent peripartum antibiotic treatment of GBS-positive mothers. Heavy GBS vaginal burden is a risk factor for both neonatal disease and colonization. Unfortunately, little is known about the host and bacterial factors that promote or permit GBS vaginal colonization. This protocol describes a technique for establishing persistent GBS vaginal colonization using a single &#946;-estradiol pre-treatment and daily sampling to determine bacterial load. It further details methods to administer additional therapies or reagents of interest and to collect vaginal lavage fluid and reproductive tract tissues. This mouse model will further the understanding of the GBS-host interaction within the vaginal environment, which will lead to potential therapeutic targets to control maternal vaginal colonization during pregnancy and to prevent transmission to the vulnerable newborn. It will also be of interest to increase our understanding of general bacterial-host interactions in the female vaginal tract.

A Murine Model of Group B Streptococcus Vaginal Colonization

The purpose of this protocol is to imitate human group B Streptococcus (GBS) vaginal colonization in a murine model. This method may be used to investigate host immune responses and bacterial factors contributing to GBS vaginal persistence, as well as to test therapeutic strategies.

Streptococcus agalactiae (group B Streptococcus, GBS), is a Gram-positive, asymptomatic colonizer of the human gastrointestinal tract and vaginal tract of ...

Histological Quantification to Determine Lung Fungal Burden in Experimental Aspergillosis

The quantification of lung fungal burden is critical for the determination of the relative levels of immune protection and fungal virulence in mouse models of pulmonary fungal infection. Although multiple methods are used to assess fungal burden, quantitative polymerase chain reaction (qPCR) of fungal DNA has emerged as a technique with several advantages over previous culture-based methods. Currently, a comprehensive assessment of lung pathology, leukocyte recruitment, fungal burden, and gene expression in mice with invasive aspergillosis (IA) necessitates the use of a significant number of experimental and control animals. Here the quantification of lung histological staining to determine fungal burden using a reduced number of animals was examined in detail. Lung sections were stained to identify fungal structures with Gomori&#39;s modified methanamine silver (GMS) staining. Images were taken from the GMS-stained sections from 4 discrete fields of each formalin-fixed paraffin-embedded lung. The GMS stained areas within each image were quantified using an image analysis program, and from this quantification, the mean percentage of stained area was determined for each sample. Using this strategy, eosinophil-deficient mice exhibited decreased fungal burden and disease with caspofungin therapy, while wild-type mice with IA did not improve with caspofungin. Similarly, fungal burden in mice lacking &#947;&#948; T cells were also improved by caspofungin, as measured by qPCR and GMS quantification. GMS quantification is therefore introduced as a method for the determination of relative lung fungal burden that may ultimately reduce the quantity of experimental animals required for comprehensive studies of invasive aspergillosis.

Here we describe a protocol to determine pulmonary fungal burden in mice with invasive aspergillosis by quantification of Gomori&#39;s modified methanamine silver staining in histological sections. Use of this method resulted in comparable results with less animals compared to assessment of fungal burden by quantitative PCR of lung fungal DNA.

The quantification of lung fungal burden is critical for the determination of the relative levels of immune protection and fungal virulence in mouse ...

Real-time Imaging and Quantification of Fungal Biofilm Development Using a Two-Phase Recirculating Flow System

In oropharyngeal candidiasis, members of the genus Candida must adhere to and grow on the oral mucosal surface while under the effects of salivary flow. While models for the growth under flow have been developed, many of these systems are expensive, or do not allow imaging while the cells are under flow. We have developed a novel apparatus that allows us to image the growth and development of Candida albicans cells under flow and in real-time. Here, we detail the protocol for the assembly and use of this flow apparatus, as well as the quantification of data that are generated. We are able to quantify the rates that the cells attach to and detach from the slide, as well as to determine a measure of the biomass on the slide over time. This system is both economical and versatile, working with many types of light microscopes, including inexpensive benchtop microscopes, and is capable of extended imaging times compared to other flow systems. Overall, this is a low-throughput system that can provide highly detailed real-time information on the biofilm growth of fungal species under flow.

We describe the assembly, operation, and cleaning of a flow apparatus designed to image fungal biofilm formation in real time while under flow. We also provide and discuss quantitative algorithms to be used on the acquired images.

In oropharyngeal candidiasis, members of the genus Candida must adhere to and grow on the oral mucosal surface while under the effects of salivary flow. ...