Progress 10/01/10 to 09/30/15
Outputs
Target Audience:
Nothing Reported
Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided?
Nothing Reported
How have the results been disseminated to communities of interest?Travis presented his data at the student research day on campus. What do you plan to do during the next reporting period to accomplish the goals?Nothing - this is the final report; project period has ended.
Impacts
What was accomplished under these goals?
White Nose Syndrome (WNS) is a fungal infection that is destroying hibernating bat populations across North America. Caused by the fungus Pseudogymnoascus destructans, infection is associated with high mortality in exposed hibernacula. There is currently no treatment available for WNS; an effective environmental treatment may prove invaluable in containing the spread of infection and as an aid in raising healthy bat populations in captivity. In addition to environmental treatments, topical treatments may be developed for use in treatment of infected individuals. This study tested the efficacy of various antifungal agents in inhibiting the growth of Pseudogymnoascus destructans by growing the fungus in cultures containing various antifungal drugs and comparing the growth between drugs. Drugs that proved successful in this study may prove to be promising candidates for in vivo testing. Work for this project was performed by Travis Beeler (V'16 Vet Student) in the summer of 2013. MATERIALS AND METHODS Three different rounds of assays were conducted for this experiment, and each round tested the efficacy of the same compounds against P. destructans. During the first two rounds of assays, we were unable to achieve substantial growth of the fungus in 96-well plates in order to test the drugs. During the final round of assays, we were able to achieve growth of the fungus in liquid culture in 96-well plates. Pseudogymnoascus destructans (Pd) Two different strains of Pd were used in this study: one North American strain (hereafter referred to as Gd) and one European strain (hereafter referred to as SJ). The Gd strain was provided by Dr. Blehert, US Fish and Wildlife Services, and the SJ strain was provided by Dr. Puechmaille, University College Dublin (6, 7). Both strains were maintained in culture on both Sabouraud and oatmeal agar plates at 8°C. Sabouraud agar plates favor the growth of hyphae while oatmeal agar plates favor the growth of conidia. Drug testing in 96-well plates Six drugs were tested for their efficacy against Pd: econazole nitrate (Sigma-Aldrich, St. Louis, MO), terbinafine HCl (Sigma-Aldrich, St. Louis, MO), lime sulfur (Vétoquinol, Ft. Worth, TX), 1-hydroxypyridine-2-thione zinc salt (Sigma-Aldrich, St. Louis, MO), itraconazole (Sigma-Aldrich, St. Louis, MO), and chlorhexidine glucuronate (Durvet, Blue Springs, MO). Drug suspensions were prepared and tested at concentrations of 10, 1, 0.1, and 0.01 µM in sterile water and 0.3% DMSO. Each drug was tested at each concentration in triplicate for inhibitory activity against both the Gd and SJ strains. Each well contained 100 µL of 3x YNBG medium, 100 µL of fungal suspension, and 100 µL of drug suspension. Fungal suspensions were prepared by harvesting fungus from oatmeal agar plates. Fungus was suspended in sterile water, and the fungal suspension was adjusted to an OD530 of 0.1 (for the first two rounds of assays) or 0.2 (for the third round of assays) using a Synergy MultiDetection plate reader (BioTek) before being added to 96-well, flat-bottom plates (Becton Dickinson Labware, Franklin Lakes, NJ). For the third round of assays, a beadbeater (Biospec Products; 20 sec maximum speed with micro beads) was used to break up clumps of fungus in the suspension. Each well contained a final concentration of 0.1% DMSO and 1x YNBG medium (or a mix at a 1:1 ratio of 3xYNBG and Sabouraud medium). Control wells consisted of 0.1% DMSO, Sabouraud medium, or water with no drug suspension. Perimeter wells on each 96-well plate were filled with water or Sabouraud medium to prevent evaporative water loss from experimental wells and monitor for contamination. Each 96-well plate was covered with a breathable membrane (Excel Scientific, Victorville, CA) in order to allow for adequate gas exchange then placed into a plastic baggie (for the first round of assays) and incubated at 8°C for two weeks. After two weeks, plates were read using the Synergy plate reader to obtain OD readings at 530 nm, and 100 µL aliquots were taken from the wells and plated onto Sabouraud agar plates for CFU counts. After an additional two weeks at 8°C, CFU counts were performed and correlated with OD530 readings obtained previously. The following table summarizes the differences between the first, second, and third rounds of assays: 1st Round 2nd Round 3rd Round Medium 3x YNBG 3x YNBG 1:1 mix of 3x YNBG and Sabouraud medium Perimeter Wells Water Sabouraud medium Sabouraud medium Fungal Suspension Clumpy, OD530=0.1 Clumpy, OD530=0.1 Homogenized, OD530=0.2 Incubation Temperature (°C) 8 8 8 Plate Cover Breathable membrane, plastic baggie Breathable membrane, no baggie Breathable membrane, no baggie Incubation Period 14 days 14 days 14 days RESULTS/DISCUSSION 1st Round of Drug Assays The OD530 data together with the data from the CFU counts suggest that there was little growth in the 96-well plates used in the first round of drug assays. Contamination was also an issue, as seen with the abnormally high absorbance values for medium-only controls. Contamination may have been an even more prevalent issue with this round of assays, but since the medium-only controls were filled with sterile water instead of Sabouraud medium, it may just be that the medium used was not rich enough to allow for visual contamination. 2nd Round of Drug Assays As with the first round of assays, the OD530 data together with the CFU counts suggest that there was little growth in the 96-well plates used in these assays. Contamination was an issue in this round of assays as well, as noted by the abnormally high absorbance values for the medium-only controls. It is more likely that contamination was visually noticed in this round of assays because the medium-only controls were filled with Sabouraud medium instead of sterile water. 3rd Round of Drug Assays The OD530 data for this round of assays was much more consistent than the highly variable values seen with the first two rounds of assays. Contamination was clearly an issue with this round of assays as well, but the absorbance values indicate that there was definite fungal growth in those plates whose medium-only control wells were not contaminated. Chlorhexidine displayed excellent inhibitory activity, itraconazole and terbinafine displayed slight inhibitory activity, pyrithione zinc and econazole displayed little to no inhibitory activity, and lime sulfur displayed no inhibitory activity. None of the drugs tested appeared to inhibit fungal growth at concentrations lower than 10 µM. Trouble Shooting Contamination was a significant problem in this project. Even when proper aseptic technique is followed, it can be difficult to avoid contamination of fungal cultures, possibly due in large part to the use of breathable membranes with the 96-well plates instead of sterile, plastic lids. Once wet, the membranes become permeable to various microbes in the environment, opening the door for contamination. The other most significant problem encountered during this project was inadequate growth of the fungus in 96-well plate cultures. The two major changes made for the third round of assays that may have allowed for adequate growth in 96-well plate cultures were doubling the amount of fungus inoculated into each well at the beginning of the assays (starting with a suspension with an OD530 of 0.2 instead of 0.1) and homogenizing the fungal suspension before use in the assays in order to circumvent the natural tendency of the fungus to clump. By working through the difficulties inherent to growing Pseudogymnoascus destructans in 96-well plate cultures, the authors hope that this project will help lay the groundwork for further work in this area. Once the fungus can be reproducibly grown in 96-well plate cultures, high-throughput in vitro screens can be performed in order to test the efficacy of a variety of pharmaceuticals against P. destructans.
Publications
|
Progress 10/01/13 to 09/30/14
Outputs
Target Audience:
Nothing Reported
Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided?
Nothing Reported
How have the results been disseminated to communities of interest?
Nothing Reported
What do you plan to do during the next reporting period to accomplish the goals?
Nothing Reported
Impacts
What was accomplished under these goals?
White Nose Syndrome (WNS) is a fungal infection that is destroying hibernating bat populations across North America. Caused by the fungus Pseudogymnoascus destructans, infection is associated with high mortality in exposed hibernacula. There is currently no treatment available for WNS; an effective environmental treatment may prove invaluable in containing the spread of infection and as an aid in raising healthy bat populations in captivity. In addition to environmental treatments, topical treatments may be developed for use in treatment of infected individuals. This study tested the efficacy of various antifungal agents in inhibiting the growth of Pseudogymnoascus destructans by growing the fungus in cultures containing various antifungal drugs and comparing the growth between drugs. Drugs that proved successful in this study may prove to be promising candidates for in vivo testing. Work for this project was performed by Travis Beeler (V'16 Vet Student) in the summer of 2013. MATERIALS AND METHODS Three different rounds of assays were conducted for this experiment over the summer, and each round tested the efficacy of the same compounds against P. destructans. During the first two rounds of assays, we were unable to achieve substantial growth of the fungus in 96-well plates in order to test the drugs. During the final round of assays, we were able to achieve growth of the fungus in liquid culture in 96-well plates. Pseudogymnoascus destructans (Pd) Two different strains of Pd were used in this study: one North American strain (hereafter referred to as Gd) and one European strain (hereafter referred to as SJ). The Gd strain was provided by Dr. Blehert, US Fish and Wildlife Services, and the SJ strain was provided by Dr. Puechmaille, University College Dublin (6, 7). Both strains were maintained in culture on both Sabouraud and oatmeal agar plates at 8°C. Sabouraud agar plates favor the growth of hyphae while oatmeal agar plates favor the growth of conidia. Drug testing in 96-well plates Six drugs were tested for their efficacy against Pd: econazole nitrate (Sigma-Aldrich, St. Louis, MO), terbinafine HCl (Sigma-Aldrich, St. Louis, MO), lime sulfur (Vétoquinol, Ft. Worth, TX), 1-hydroxypyridine-2-thione zinc salt (Sigma-Aldrich, St. Louis, MO), itraconazole (Sigma-Aldrich, St. Louis, MO), and chlorhexidine glucuronate (Durvet, Blue Springs, MO). Drug suspensions were prepared and tested at concentrations of 10, 1, 0.1, and 0.01 µM in sterile water and 0.3% DMSO. Each drug was tested at each concentration in triplicate for inhibitory activity against both the Gd and SJ strains. Each well contained 100µL of 3x YNBG medium, 100µL of fungal suspension, and 100µL of drug suspension. Fungal suspensions were prepared by harvesting fungus from oatmeal agar plates. Fungus was suspended in sterile water, and the fungal suspension was adjusted to an OD530 of 0.1 (for the first two rounds of assays) or 0.2 (for the third round of assays) using a Synergy MultiDetection plate reader (BioTek) before being added to 96-well, flat-bottom plates (Becton Dickinson Labware, Franklin Lakes, NJ). For the third round of assays, a beadbeater (Biospec Products; 20 sec maximum speed with micro beads) was used to break up clumps of fungus in the suspension. Each well contained a final concentration of 0.1% DMSO and 1x YNBG medium (or a mix at a 1:1 ratio of 3xYNBG and Sabouraud medium). Control wells consisted of 0.1% DMSO, Sabouraud medium, or water with no drug suspension. Perimeter wells on each 96-well plate were filled with water or Sabouraud medium to prevent evaporative water loss from experimental wells and monitor for contamination. Each 96-well plate was covered with a breathable membrane (Excel Scientific, Victorville, CA) in order to allow for adequate gas exchange then placed into a plastic baggie (for the first round of assays) and incubated at 8°C for two weeks. After two weeks, plates were read using the Synergy plate reader to obtain OD readings at 530nm, and 100µL aliquots were taken from the wells and plated onto Sabouraud agar plates for CFU counts. After an additional two weeks at 8°C, CFU counts were performed and correlated with OD530 readings obtained previously. The following table summarizes the differences between the first, second, and third rounds of assays: 1st Round 2nd Round 3rd Round Medium 3x YNBG 3x YNBG 1:1 mix of 3x YNBG and Sabouraud medium Perimeter Wells Water Sabouraud medium Sabouraud medium Fungal Suspension Clumpy, OD530=0.1 Clumpy, OD530=0.1 Homogenized, OD530=0.2 Incubation Temperature (°C) 8 8 8 Plate Cover Breathable membrane, plastic baggie Breathable membrane, no baggie Breathable membrane, no baggie Incubation Period 14 days 14 days 14 days RESULTS/DISCUSSION 1st Round of Drug Assays The OD530 data together with the data from the CFU counts suggest that there was little growth in the 96-well plates used in the first round of drug assays. Contamination was also an issue, as seen with the abnormally high absorbance values for medium-only controls. Contamination may have been an even more prevalent issue with this round of assays, but since the medium-only controls were filled with sterile water instead of Sabouraud medium, it may just be that the medium used was not rich enough to notice the contamination. 2nd Round of Drug Assays As with the first round of assays, the OD530 data together with the CFU counts suggest that there was little growth in the 96-well plates used in these assays. Contamination was an issue in this round of assays as well, as noted by the abnormally high absorbance values for the medium-only controls. It is more likely that contamination was noticed in this round of assays than in the first since the medium-only controls were filled with Sabouraud medium instead of sterile water. 3rd Round of Drug Assays The OD530 data for this round of assays was much more consistent than the highly variable values seen with the first two rounds of assays. Contamination was clearly an issue with this round of assays as well, but the absorbance values indicate that there was definite fungal growth in those plates whose medium-only control wells were not contaminated. Chlorhexidine displays excellent inhibitory activity, itraconazole and terbinafine display slight inhibitory activity, pyrithione zinc and econazole display little to no inhibitory activity, and lime sulfur displays no inhibitory activity. None of the drugs tested appeared to inhibit fungal growth at concentrations lower than 10µM. Trouble Shooting Contamination was a significant problem in this project. Even when proper aseptic technique is followed, it can be difficult to avoid contamination of fungal cultures, possibly due in large part to the use of breathable membranes with the 96-well plates instead of sterile, plastic lids. Once wet, the membranes become permeable to various microbes in the environment, opening the door for contamination. The other most significant problem encountered during this project was inadequate growth of the fungus in 96-well plate cultures. The two major changes made for the third round of assays that may have allowed for adequate growth in 96-well plate cultures were doubling the amount of fungus inoculated into each well at the beginning of the assays (starting with a suspension with an OD530 of 0.2 instead of 0.1) and homogenizing the fungal suspension before use in the assays in order to circumvent the natural tendency of the fungus to clump. By working through the difficulties inherent to growing Pseudogymnoascus destructans in 96-well plate cultures, the authors hope that this project will help lay the groundwork for further work in this area. Once the fungus can be reproducibly grown in 96-well plate cultures, high-throughput in vitro screens can be performed in order to test the efficacy of a variety of pharmaceuticals against P. destructans
Publications
|
Progress 10/01/12 to 09/30/13
Outputs
Target Audience: TCSVM students, staff and faculty Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided?
Nothing Reported
How have the results been disseminated to communities of interest? Research was orally presented at the TCSVM Veterinary Students Summer Research Day in Fall 2013 What do you plan to do during the next reporting period to accomplish the goals? This was a summer student project- ended in August 2013.
Impacts
What was accomplished under these goals?
White Nose Syndrome (WNS) is a fungal infection that is destroying hibernating bat populations across North America. Caused by the fungus Pseudogymnoascus destructans, infection is associated with high mortality in exposed hibernacula. There is currently no treatment available for WNS; an effective environmental treatment may prove invaluable in containing the spread of infection and as an aid in raising healthy bat populations in captivity. In addition to environmental treatments, topical treatments may be developed for use in treatment of infected individuals. This study tested the efficacy of various antifungal agents in inhibiting the growth of Pseudogymnoascus destructans by growing the fungus in cultures containing various antifungal drugs and comparing the growth between drugs. Drugs that proved successful in this study may prove to be promising candidates for in vivo testing. MATERIALS AND METHODS Three different rounds of assays were conducted for this experiment over the summer, and each round tested the efficacy of the same compounds against P. destructans. During the first two rounds of assays, we were unable to achieve substantial growth of the fungus in 96-well plates in order to test the drugs. During the final round of assays, we were able to achieve growth of the fungus in liquid culture in 96-well plates. Pseudogymnoascus destructans (Pd) Two different strains of Pd were used in this study: one North American strain (hereafter referred to as Gd) and one European strain (hereafter referred to as SJ). The Gd strain was provided by Dr. Blehert, US Fish and Wildlife Services, and the SJ strain was provided by Dr. Puechmaille, University College Dublin (6, 7). Both strains were maintained in culture on both Sabouraud and oatmeal agar plates at 8°C. Sabouraud agar plates favor the growth of hyphae while oatmeal agar plates favor the growth of conidia. Drug testing in 96-well plates Six drugs were tested for their efficacy against Pd: econazole nitrate (Sigma-Aldrich, St. Louis, MO), terbinafine HCl (Sigma-Aldrich, St. Louis, MO), lime sulfur (Vétoquinol, Ft. Worth, TX), 1-hydroxypyridine-2-thione zinc salt (Sigma-Aldrich, St. Louis, MO), itraconazole (Sigma-Aldrich, St. Louis, MO), and chlorhexidine glucuronate (Durvet, Blue Springs, MO). Drug suspensions were prepared and tested at concentrations of 10, 1, 0.1, and 0.01 µM in sterile water and 0.3% DMSO. Each drug was tested at each concentration in triplicate for inhibitory activity against both the Gd and SJ strains. Each well contained 100µL of 3x YNBG medium, 100µL of fungal suspension, and 100µL of drug suspension. Fungal suspensions were prepared by harvesting fungus from oatmeal agar plates. Fungus was suspended in sterile water, and the fungal suspension was adjusted to an OD530 of 0.1 (for the first two rounds of assays) or 0.2 (for the third round of assays) using a Synergy MultiDetection plate reader (BioTek) before being added to 96-well, flat-bottom plates (Becton Dickinson Labware, Franklin Lakes, NJ). For the third round of assays, a beadbeater (Biospec Products; 20 sec maximum speed with micro beads) was used to break up clumps of fungus in the suspension. Each well contained a final concentration of 0.1% DMSO and 1x YNBG medium (or a mix at a 1:1 ratio of 3xYNBG and Sabouraud medium). Control wells consisted of 0.1% DMSO, Sabouraud medium, or water with no drug suspension. Perimeter wells on each 96-well plate were filled with water or Sabouraud medium to prevent evaporative water loss from experimental wells and monitor for contamination. Each 96-well plate was covered with a breathable membrane (Excel Scientific, Victorville, CA) in order to allow for adequate gas exchange then placed into a plastic baggie (for the first round of assays) and incubated at 8°C for two weeks. After two weeks, plates were read using the Synergy plate reader to obtain OD readings at 530nm, and 100µL aliquots were taken from the wells and plated onto Sabouraud agar plates for CFU counts. After an additional two weeks at 8°C, CFU counts were performed and correlated with OD530 readings obtained previously. RESULTS/DISCUSSION 1st Round of Drug Assays The OD530 data together with the data from the CFU counts suggest that there was little growth in the 96-well plates used in the first round of drug assays. Contamination was also an issue, as seen with the abnormally high absorbance values for medium-only controls. Contamination may have been an even more prevalent issue with this round of assays, but since the medium-only controls were filled with sterile water instead of Sabouraud medium, it may just be that the medium used was not rich enough to notice the contamination. 2nd Round of Drug Assays As with the first round of assays, the OD530 data together with the CFU counts suggest that there was little growth in the 96-well plates used in these assays. Contamination was an issue in this round of assays as well, as noted by the abnormally high absorbance values for the medium-only controls. It is more likely that contamination was noticed in this round of assays than in the first since the medium-only controls were filled with Sabouraud medium instead of sterile water. 3rd Round of Drug Assays The OD530 data for this round of assays was much more consistent than the highly variable values seen with the first two rounds of assays. Contamination was clearly an issue with this round of assays as well, but the absorbance values indicate that there was definite fungal growth in those plates whose medium-only control wells were not contaminated. Chlorhexidine displays excellent inhibitory activity, itraconazole and terbinafine display slight inhibitory activity, pyrithione zinc and econazole display little to no inhibitory activity, and lime sulfur displays no inhibitory activity. None of the drugs tested appeared to inhibit fungal growth at concentrations lower than 10µM. Trouble Shooting Contamination was a significant problem in this project. Even when proper aseptic technique is followed, it can be difficult to avoid contamination of fungal cultures, possibly due in large part to the use of breathable membranes with the 96-well plates instead of sterile, plastic lids. Once wet, the membranes become permeable to various microbes in the environment, opening the door for contamination. The other most significant problem encountered during this project was inadequate growth of the fungus in 96-well plate cultures. The two major changes made for the third round of assays that may have allowed for adequate growth in 96-well plate cultures were doubling the amount of fungus inoculated into each well at the beginning of the assays (starting with a suspension with an OD530 of 0.2 instead of 0.1) and homogenizing the fungal suspension before use in the assays in order to circumvent the natural tendency of the fungus to clump. By working through the difficulties inherent to growing Pseudogymnoascus destructans in 96-well plate cultures, the authors hope that this project will help lay the groundwork for further work in this area. Once the fungus can be reproducibly grown in 96-well plate cultures, high-throughput in vitro screens can be performed in order to test the efficacy of a variety of pharmaceuticals against P. destructans.
Publications
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2013
Citation:
Travis Beeler (V'16 Vet Student) internal publication and presentation titled "IN VITRO EVALUATION OF ANTIFUNGAL COMPOUNDS AGAINST PSEUDOGYMNOASCUS DESTRUCTANS, THE CAUSAL AGENT OF WHITE NOSE SYNDROME IN BATS" - Veterinary Student Summer Project Research Day at TCSVM in 2013.
|
Progress 01/01/12 to 09/30/12
Outputs
Target Audience: TCSVM students, staff and faculty. Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided?
Nothing Reported
How have the results been disseminated to communities of interest? Research was orally presented at the TCSVM Veterinary Students Summer Research Day in Fall 2012 What do you plan to do during the next reporting period to accomplish the goals?
Nothing Reported
Impacts
What was accomplished under these goals?
White Nose Syndrome is a novel fungal infection that is decimating hibernating bat populations in the United States. There are no current treatments for the disease and it is very likely impossible to contain since it is spread by bats as they fly to different hibernacula. It may become necessary to maintain captive colonies of bats to prevent extinction. Although some in vitro studies have been performed to obtain potential drug candidates, the development of an improved in vitro assay for drug testing would be beneficial in finding a treatment. Thus, the aim of this research was to technology-transfer a published 96-well assay and to test the assay. Materials and Methods. Laboratory Strains of Gd. Two Gd isolates were evaluated in liquid culture antifungal testing, a USA isolate (provided by Dr. Blehert, US Fish and Wildlife Services) that was sequenced by the Broad Institute and a European isolate (provided by Dr. Puechmaille, University College Dublin). Gd was plated on both Sabouraud agar and oatmeal agar (60 g/L oatmeal, 15% agar) and incubated at 12-13°C for 10 days. Colonies were then scraped off the plates and added to sterile water. Microscopy was used to analyze the whether the Gd had grown as hyphae or conidia (spores). Gd conidia from oatmeal plates were also grown in liquid culture under the same conditions, using either Gd medium (40 g/L dextrose, 10 g/L peptone, pH to 6.8-7) or 2% yeast nitrogen base containing amino acids and 4% glucose (YNBG). Liquid Culture Antifungal Susceptibility Testing Gd was grown on oatmeal agar for 10 days. Both a US strain and an European Gd strain were tested. Suspensions of Gd (0.1 OD530) were incubated with different concentrations of the test drugs (originally 10, 1, 0.1, 0.01, 0.001 and 0.0001 μM, made up in 0.1% DMSO) for 10-14 days in 96-well plates containing 4% glucose 2% yeast nitrogen base plus amino acids (YNBG). Each well contained 100 μL YNBG medium, 100 μL of the 0.1 OD530 Gd solution, and 100 μL of the drug in question. The drugs tested were econazole nitrate, itraconazole, zinc pyrithione, terbinafine, lime sulfur and chlorhexidine digluconate (Table 1). Both sterile water and sterile 0.1% DMSO control wells were also used. Each drug concentration was tested twice per plate on two plates. At the 10-14 day time point, the OD530 of each of the wells was measured using a Synergy H4 Multi-Detection Plate Reader (BioTek) to evaluate levels of inhibition by the drugs. The wells containing various concentrations of each drug were also plated to provide quantitative results and prove viability of the Gd. 100 μL of solution from each of the wells were plated on Sabouraud agar. Two samples of each drug concentration were plated, one from each microwell. After two weeks of growth, the colonies were counted and recorded. This assay was repeated several times. Testing with a Breathable Sealing Membrane on the 96 well Plate. Gd (0.1 OD530) was added to the wells of a 96-well plate, mimicking the liquid culture antifungal testing. 100 μL of the Gd was added to 100 μL YNBG and 100 μL of sterile water. However, instead of the usual plastic cover for the 96 well plate, a sterile breathable sealing membrane was placed over the top of the wells instead. Results Liquid Culture Antifungal Susceptibility Testing. Gd from oatmeal agar plates were harvested, resuspended in sterile water and used in the assay. YNBG medium was used in the assay. Measuring inhibition of growth by measuring OD530 proved unsuccessful, as the spectrophotometer was not sensitive enough to differentiate between the quantities of Gd in the wells. Although replicating the conditions used by Chaturvedi et al. as closely as possible, this experiment was unable to obtain the same level of Gd growth that allowed them to measure a change in OD530. However, plating of the cultures on Sabouroud agar allowed for a quantification of the effects of the drug. For the US strain of Gd, wells containing 10 μM itraconazole, econazole, lime sulfur and chlorhexidine showed very little or no Gd growth on plates. Pyrithione zinc showed some reduction, whereas terbinafine wells had similar colony numbers to control wells. DMSO did not appear to affect colony numbers, as seen by comparing sterile water controls to 0.1% DMSO controls. These results were similar for the European Gd strain, except that chlorhexidine showed complete inhibition of Gd growth at 1 μM as well as 10 μM. These results could not be analyzed statistically because of the length of time it took to determine appropriate concentrations. Testing with a Breathable Sealing Membrane on the 96 well Plate. After 10 days, the wells containing Gd under the breathable sealing membrane showed visible fungal growth. OD530 measurements showed values ranging from 0.168 to 0.307, which were much higher than the ODs obtained with the usual plastic cover over the microwells. This method is a potential improvement that can be used when testing antifungals in 96 well plates in the future. Discussion We focused on developing the liquid culture antifungal testing, as performed by Chaturvedi et al. To test drugs against the spore form of the fungus, called conidia. Thus, we chose to grow the Gd on oatmeal agar plates because the majority fungal form is conidia. We followed the protocol of Chaturvedi et al. almost exactly, except Gd was incubated at 12-13 degrees Celsius rather than 6 and 15 degrees, and the cultures were incubated for two weeks instead of ten days. We expected that these slight changes would not drastically impact results. However, even our control wells with no drugs present had very little Gd growth over the two-week incubation period. The spectrophotometer was unable to differentiate between the treated wells and the control wells. Although the medium used (2% yeast nitrogen base 4% glucose) is a minimal medium, Chaturvedi et al. demonstrated significant growth of Gd over their 10-day incubation. The picture included in their study shows microwells with visible fungal growth, whereas our wells remained clear. As a test, we mimicked the setup of the microwell experiment but used a sterile breathable sealing membrane instead of the normal top. After 10 days of incubation at 12-13°C, there was visible Gd growth and a significant increase in OD530. It is possible that repeating our studies with these breathable membranes would provide better results. Since we could not use spectrophotometry to differentiate between the treated and non-treated wells, the cultures from the various wells were plated on Sabouraud plates to quantitate the number of Gd. After a two-week incubation period, the colonies were counted. Our results suggest that some drugs inhibited Gd growth whereas others did not. For both the US and the European strains, econazole, itraconazole, lime sulfur and chlorhexidine showed a drastic reduction in Gd at 10 µM concentration. For the European strain, 1 µM chlorhexidine inhibited Gd. These results suggest that future studies should investigate concentrations around 10 µM. Due to time constraints and optimizing of this assay, we were unable to perform enough replicate assays to perform statistical analysis. Future experiments following this procedure should include testing each drug concentration in duplicate or triplicate as well as duplicate plates.
Publications
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2012
Citation:
Brittany Pereira (V'15 Vet Student) internal publication and presentation titled " Development of an Infective Hair Model for Testing Antifungal Drugs Against Geomyces Destructans" - Veterinary Student Summer Project Research Day at TCSVM in 2012
|
Progress 01/01/11 to 12/31/11
Outputs
OUTPUTS: Geomyces destructans is the causal agent of the disease affecting hibernating bats named White nose syndrome. This high mortality disease has caused the loss up to 99% of hibernating bats in New England. The gold standard for disease diagnosis is made through histopathology of bat wings, a post-mortem only diagnosis. There is a need for anti-mortem diagnostic techniques especially for one that could be utilized in the field easily. In an effort to develop alternative diagnostic techniques, we have generated 5 separate monoclonal antibodies. We have characterized these monoclonal antibodies as to type. All 5 bind specifically to Geomyces destructans under experimental conditions. These antibodies will be useful for diagnostic and research purposes once they are further developed for those purposes. More monoclonals are being developed which bind to surface proteins of the Gd organism, increasing the likelihood of usefulness in diagnostic assays and other techniques. PARTICIPANTS: Nothing significant to report during this reporting period. TARGET AUDIENCES: Nothing significant to report during this reporting period. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.
Impacts
Monoclonal antibodies that bind pathogens are an essential tool for research and diagnostic purposes. Geomyces destructans is most likely present for indefinite periods in the environment where bats hibernate. The ecology of this pathogen is not well understood because it is very hard to isolate from environmental samples. Additionally, as the disease spreads across the continent, new species of bats are becoming infected with the disease, and histopathology is not always a reliable means for identifying the infection in different species associated with different hibernation conditions. Monoclonal antibodies are going to be an essential tool to diagnose the disease both in bats, and in environmental samples. PCR is a competing method to diagnose infection, however developing a specific PCR probe has proved elusive even 5 years into research in this area. One reason for the failure of PCR is that the Gd genome is huge, and there are hundreds of similar Geomyces (nearly all undescribed) present in the environment. Monoclonal antibody techniques are likely to advance diagnostic methods, and research into the ecology and epidemiology of the disease.
Publications
- No publications reported this period
|
|