I would like to thank C.L. Richards-Zawacki for help conceptualizing the experimental design and assisting with materials, A. Pessier for help establishing a treatment regimen and necropsy analyses, M.W.H. Chatfield, M.J. Robak, K. Tasker and D. Lenger for help in the laboratory and support and M. McFadden and P. Harlow at the Taronga Zoo for use of their space during filming. The project was partially funded by Louisiana Department of Wildlife and Fisheries through the Louisiana Environmental Education Commission grant awarded to L.A. Brannelly and by Life Technologies.

Ethics Approval: This study and its methods were approved by Tulane University&#39;s Animal Use and Care Committee (protocol no. 0407).
1. Testing for Bd Infection
<ol>
	<li>Swabbing for Bd
	<ol>
		<li>Swab animals with a sterile cotton swab five times on the ventrum, five times on each thigh, each side, and each limb for a total of 45 strokes. Swabbing is a noninvasive method of sampling for Bd on amphibian skin.</li>
		<li>Gently rotate the swab during and between strokes to ensure the greatest amount of DNA is gathered on the swab.</li>
		<li>Store swabs in 1.5 ml microtubes at -20 &#176;C.</li>
	</ol>
	</li>
	<li>DNA Extraction and qPCR assay
	<ol>
		<li>Extract genomic DNA from the swabs using a commercially available DNA isolation kit, in accordance with the instructions for animal tissue.</li>
		<li>Analyze the extracted DNA using quantitative real time PCR (qPCR) following Boyle et al.17, with the following modifications:</li>
		<li>Dilute DNA extraction samples 1:10 with double deionized water.</li>
		<li>Add 0.7 &#181;l of bovine serum albumin (BSA) to prevent PCR inhibition.</li>
		<li>Include an internal positive control in each sample to ensure PCR inhibition was not affecting results.</li>
		<li>Run samples with a positive and negative control and a series of dilution standards to estimate zoospore (infection) load.</li>
	</ol>
	</li>
</ol>
2. Inoculation
<ol>
	<li>Culture Bd
	<ol>
		<li>Prepare tryptone broth by adding 10 g tryptone to 1 L reverse osmosis or deionized water, autoclaved (121 &#176;C for 40 min) and allowed to cool. Once cooled to 23 &#176;C, add 1 ml penicillin-streptomycin.</li>
		<li>Grow Bd strain (JEL411 from J. Longcore, isolated from Guabal, Panama from a Phyllomedusa lemur) in a tryptone broth for seven days at 23 &#176;C.</li>
	</ol>
	</li>
	<li>Transfer the broth culture to tryptone and agar plates.
	<ol>
		<li>To make plates, add 10 g tryptone and 10 g agar to 1 L reverse osmosis or deionized water and autoclave (121 &#176;C for 40 min).</li>
		<li>Once the autoclaved mixture is cool enough to touch, but before it congeals, pour into Petri dishes, 1/4-1/3 full, in a laminar flow hood.</li>
		<li>When agar has congealed and cooled completely, add 0.5 ml Bd broth to each plate and spread evenly around the plate. Allow to dry.</li>
		<li>Once the plates are inoculated with Bd zoospores, seal plates with Parafilm.</li>
		<li>Allow plates to incubate at 23 &#176;C for five to seven days. Check zoospore movement to ensure viability before inoculation.</li>
	</ol>
	</li>
	<li>Flood plates for zoospore inoculation solution
	<ol>
		<li>After the five to seven days of Bd incubation and growth, flood each plate with 5 ml of aged tap water and allow zoospores to enter the media for 10 min.</li>
		<li>Pour the zoospore suspension into a portable container.</li>
		<li>Estimate zoospore concentration with a hemocytometer.</li>
		<li>Dilute mixture to a concentration of 1 x 106&#160;per&#160;3 ml with aged tap water.</li>
	</ol>
	</li>
	<li>Inoculate animals
	<ol>
		<li>In individual 50 ml containers, inoculate each animal by pouring 3 ml inoculum mixture over its ventrum. Allow extra inoculum to drip into the base of the inoculation container.</li>
		<li>Each animal will remain in inoculation container for 24 hr to ensure infection.</li>
		<li>After 24 hr of inoculation, return animals to individual disinfected terraria.</li>
		<li>Allow infection to build for two weeks. Confirm infection with swab and qPCR results, and begin treatment.</li>
		<li>For Bd&#173;-negative controls, flood Bd-free agar plates with 5 ml aged tap water, and mock inoculate the animals using the same methods as above.</li>
	</ol>
	</li>
</ol>
3. Treatment Regimen
<ol>
	<li>Prepare treatment bath
	<ol>
		<li>Add 2.50 ml aqueous itraconazole (1% Sporanox Oral Solution) to 1 L Amphibian Ringer&#39;s Solution (0.0025% itraconazole - the lowest effective treatment concentration).
		<ol>
			<li>Amphibian Ringer&#39;s Solution recipe: 1 L reverse osmosis or deionized water, 6.6 g sodium chloride, 0.15 g potassium chloride, 0.15 g calcium chloride, and 0.2 g sodium bicarbonate. Autoclave solution (121 &#176;C for 40 min) and allow to cool to room temperature.</li>
			<li>For 0.01% itraconazole treatment concentration, add 10.00 ml aqueous itraconazole (1% Sporanox Oral Solution) to 1 L Amphibian Ringer&#39;s Solution</li>
			<li>For 0.005% itraconazole treatment concentration, add 5.00 ml aqueous itraconazole (1% Sporanox Oral Solution) to 1 L Amphibian Ringer&#39;s Solution.</li>
		</ol>
		</li>
		<li>For Bd-positive and Bd-negative controls, bathe animals in Amphibian Ringer&#39;s Solution with no itraconazole.</li>
	</ol>
	</li>
	<li>Treatment
	<ol>
		<li>Place individual amphibians in disinfected 50 ml containers with 35 ml of the treatment solution.</li>
		<li>Ensure animals are fully covered for five minutes by gently swirling the container.</li>
		<li>Return animals to disinfected terraria. Repeat for six days.</li>
	</ol>
	</li>
	<li>Ensure treatment success
	<ol>
		<li>Test animals for Bd infection on the day treatment commences.</li>
		<li>Test animals for Bd infection one week after treatment finishes, and once a week for four weeks to ensure infection has cleared.</li>
	</ol>
	</li>
</ol>
4. Bd as a Biohazard
<ol>
	<li>Disinfect individual terraria weekly.
	<ol>
		<li>Bathe the terraria and treatment containers in a 10% bleach solution bath.</li>
		<li>Rinse each bleached container twice with running water.</li>
		<li>Allow containers to dry for 24 hr before reuse.</li>
	</ol>
	</li>
	<li>Wear clean nitrile gloves when handling animals to prevent cross contamination of Bd pathogen. Change gloves between handling each animal.</li>
	<li>Disinfect all liquid waste prior to disposal by bringing the solution to 10% bleach.</li>
	<li>Autoclave all solid waste that is potentially exposed to Bd prior to disposal.</li>
</ol>

<ol>
	<li>Stuart, S. N., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Status+and+trends+of+amphibian+declines+and+extinctions+worldwide.">Status and trends of amphibian declines and extinctions worldwide.</a> Science. 306 (5702), 1783-1786 (2004).</li><li>Skerratt, L. F., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Spread+of+chytridiomycosis+has+caused+the+rapid+global+decline+and+extinction+of+frogs.">Spread of chytridiomycosis has caused the rapid global decline and extinction of frogs.</a> EcoHealth. 4, 125-134 (2007).</li><li>Wake, D. B., Vredenburg, V. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Colloquium+paper:+are+we+in+the+midst+of+the+sixth+mass+extinction?+A+view+from+the+world+of+amphibians.">Colloquium paper: are we in the midst of the sixth mass extinction? A view from the world of amphibians.</a> PNAS. 105, 11466-11473 (2008).</li><li>Voyles, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Electrolyte+depletion+and+osmotic+imbalance+in+amphibians+with+chytridiomycosis.">Electrolyte depletion and osmotic imbalance in amphibians with chytridiomycosis.</a> Dis. Aquat. Org. 77 (2), 113-118 (2007).</li><li>Ramsey, J. 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P., Mendelson, 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> A Manual For Control of Infectious Diseases in Amphibian Survival Assurance Colonies and Reintroduction Programs. , (2009).</li><li>Page, S. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chloramphenicol+1.+Hazards+of+use+and+the+current+regulatory+environment.">Chloramphenicol 1. Hazards of use and the current regulatory environment.</a> Aus. Vet. J. 68 (1), 1-2 (1991).</li><li>Yoon, H. -. J., Seo, M. H., Kim, J. -. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Pharmaceutical formulations for itraconazole. , (2005).</li><li>Garner, T. W. J., Garcia, G., Carroll, B., Fisher, M. 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The author declares no competing financial interests.

Using this protocol we were able to safely and effectively reduce the treatment dose and time of itraconazole treatment for Bd in amphibians. The lowest treatment dose and time (0.0025% for six days) was a successful treatment for subclinically infected I. nebulifer. The effectiveness of both a lower dose and treatment time demonstrate the need for clinical treatment trials for Bd infection treatments, as there are currently no safe and effective treatments to be used for all species and age cl...

Amphibians are currently experiencing the greatest decline in biodiversity of all vertebrate taxa1. Chytridiomycosis, a skin disease caused by the chytrid fungal pathogen Batrachochytrium dendrobatidis (Bd), is one of the major causes of these dramatic declines2. Bd is the worst pathogen on record for causing biodiversity loss: it infects over 600 species of amphibians and has caused declines in over 300 species, of which as many as 200 species have become critically endangered or extinct2,3. The amphibian chytrid fungus is not host specific and it is unclear what qualities of the host allow for Bd infection to progress causing severe disease. Chytridiomycosis causes death by disrupting ion channels in the skin, resulting in electrolyte loss and heart failure4. Also, Bd supernatant (containing no Bd zoospores) causes an immune response in tadpoles5 leading researchers to believe Bd produces a toxic by-product6.
Because many species are rapidly declining, captive assurance colonies and breeding programs are important conservation measures, and may be the only lifeline for some species. Establishing and maintaining disease free colonies is a challenge for amphibian management facilities. The current chemotherapeutic methods for treating Bd infections, although effective, can be harmful to the animals being treated, and no single treatment method has been successful across species and age classes of amphibians7.
Few clinical trials of treatments for Bd infection have been performed7. The three most commonly used methods of treatment are heat therapy, chloramphenicol and itraconazole. Heat therapy is a nonchemotherapeutic treatment option that appears to have few side effects, can be used on multiple life stages, and is generally effective. In a treatment trial, Chatfield and Richards-Zawacki8 found that 96% of the treated animals had cleared infections after being housed at 30 &#176;C for 10 days. Although this trial was not 100% successful, it provides a baseline for others to assess heat therapy for their own species. Heat therapy, although effective for many species, is impractical for many alpine species that cannot remain at high temperatures for an extended period of time. Chloramphenicol, an antibacterial, has been used successfully to treat Bd in many species, and inhibits fungal growth in vitro. As an antibacterial, the chloramphenicol does not attack the fungus specifically but has been known to cause apoptosis in eukaryotic cells9. It has been speculated that chloramphenicol therapy causes apoptosis in the skin, killing intracellular sporangia, although the exact mechanism of action is unknown. Although no clinical trial has been conducted, and therefore dose and treatment time have not been assessed, current treatment protocols typically require submersion for two to four weeks10,11, making this treatment&#160;impractical for terrestrial amphibians. Chloramphenicol has also been restricted in some countries for veterinary use because human exposure has been associated with a rare form of aplastic anemia12.
Itraconazole, an azole antifungal, is the most widely used for treatment of Bd infection in amphibians in captivity. The most widely used protocol is bathing animals in a 0.01% itraconazole solution for five minutes a day for 11 consecutive days. This protocol was derived from standards used for mammalian administration, and, while effective for amphibians, it is not based on clinical trials in amphibians11,13. This protocol can cause negative side effects in certain species, especially in the genus Rana and in tadpoles and recent metamorphs11. Tadpole treatment has been successful using a much lower concentration (0.0005%) of itraconazole, but was associated with depigmentation14. This same concentration was trialed on recent metamorphs and found to be unsuccessful at treating infection15. Because there is risk of negative side effects with itraconazole treatment, some practitioners have used one half the usual concentration (0.005%) for 11 consecutive days and found this to be successful in curing Bd infections16.
The present study is a summary of a clinical treatment trial using itraconazole to treat Bd infection15. The protocol describes an attempt to find the lowest dose and treatment time that is both safe and effective for treating Bd infected animals with itraconazole. Animals used in this study were recent metamorphs of gulf coast toads, Incilius nebulifer.

<table border="0" cellpadding="0" cellspacing="0" width="503">
	<tbody>
		<tr height="42">
			<td height="42" style="height:42px;width:179px;">0.1% Aqueous Itraconazole, Sporonox</td>
			<td style="width:147px;">Janssen Pharmaceutica Inc.</td>
			<td style="width:177px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:179px;">NaCl</td>
			<td style="width:147px;">Fisher Scientific</td>
			<td style="width:177px;">S640-500</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:179px;">KCl</td>
			<td style="width:147px;">Fisher Scientific</td>
			<td style="width:177px;">BP366-1</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:179px;">CaCl2</td>
			<td style="width:147px;">Fisher Scientific</td>
			<td style="width:177px;">BP510-100</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:179px;">NaHCO3</td>
			<td style="width:147px;">Fisher Scientific</td>
			<td style="width:177px;">BP328-500</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:179px;">Sterile cotton swabs</td>
			<td style="width:147px;">Medical and Wire Equipment</td>
			<td style="width:177px;">MW113</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:179px;">1.5 ml micro tubes</td>
			<td style="width:147px;">Sarstedt</td>
			<td style="width:177px;">73.703.217</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:179px;">Qiagen DNEasy Blood and Tissue Kit</td>
			<td style="width:147px;">Qiagen</td>
			<td style="width:177px;">69581</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:179px;">7500 Real-time PCR</td>
			<td style="width:147px;">Applied Biosystems</td>
			<td style="width:177px;">4351104</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:179px;">VIC IPC</td>
			<td style="width:147px;">Applied Biosystems</td>
			<td style="width:177px;">4308323</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:179px;">BSA</td>
			<td style="width:147px;">Applied Biosystems</td>
			<td style="width:177px;">AM2618</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:179px;">Agar</td>
			<td style="width:147px;">Invitrogen</td>
			<td style="width:177px;">30391023</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:179px;">Tryptone</td>
			<td style="width:147px;">Invitrogen</td>
			<td style="width:177px;">Q10029</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:179px;">Penicillin-streptomycin</td>
			<td style="width:147px;">Invitrogen</td>
			<td style="width:177px;">15070-063</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:179px;">Petri Dish</td>
			<td style="width:147px;">Sigma Aldrich</td>
			<td style="width:177px;">CLS430589</td>
		</tr>
	</tbody>
</table>

reduced itraconazole concentration and durations are successful in treating batrachochytrium dendrobatidis infection in amphibians

Amphibians are experiencing the greatest decline of any vertebrate class and a leading cause of these declines is a fungal pathogen, Batrachochytrium dendrobatidis (Bd), which causes the disease chytridiomycosis. Captive assurance colonies are important worldwide for threatened amphibian species and may be the only lifeline for those in critical threat of extinction. Maintaining disease free colonies is a priority of captive managers, yet safe and effective treatments for all species and across life stages have not been identified. The most widely used chemotherapeutic treatment is itraconazole, although the dosage commonly used can be harmful to some individuals and species. We performed a clinical treatment trial to assess whether a lower and safer but effective dose of itraconazole could be found to cure Bd infections. We found that by reducing the treatment concentration from 0.01-0.0025% and reducing the treatment duration from 11-6 days of 5 min baths, frogs could be cured of Bd infection with fewer side effects and less treatment-associated mortality.

Itraconazole treatment was considered successful if all animals were Bd-negative four weeks after treatment ended. Before treatment commenced, two weeks after inoculation, all Bd-inoculated animals were infected, with a mean zoospore load of 1,121.7 (&#177;17.34), while four weeks after treatment ended, the Bd-positive control animals had a mean zoospore load of 16,765.12 (&#177;14643.08).
Treatment of Incilius nebulifer (n = 15) was successful using baths i...

Watch this Scientific Journal Video about Reduced Itraconazole Concentration and Durations Are Successful in Treating Batrachochytrium dendrobatidis Infection in Amphibians at JoVE.com

Reduced Itraconazole Concentration and Durations Are Successful in Treating Batrachochytrium dendrobatidis Infection in Amphibians

Batrachochytrium dendrobatidis infection in amphibians can be eliminated using a reduced concentration (0.0025%) and shorter duration (five&#160;minute baths for six days) of itraconazole than is generally used. Less mortality and fewer negative side effects were observed using this modified treatment protocol.

Reduced Itraconazole Concentration and Durations Are Successful in Treating Batrachochytrium dendrobatidis Infection in Amphibians

Amphibians are experiencing the greatest decline of any vertebrate class and a leading cause of these declines is a fungal pathogen, Batrachochytrium ...

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Immunology and Infection

11.5K Views.  James Cook University.  Batrachochytrium dendrobatidis инфекции у земноводных могут быть устранены с помощью пониженную концентрацию (0,0025%) и короткую продолжительность (пять минут ванны для шести дней) итраконазола, чем обычно используется. Менее смертность и меньше негативных побочных эфф...

Video: Reduced Itraconazole Concentration and Durations Are Successful in Treating Batrachochytrium dendrobatidis Infection in Amphibians

Visualizing Dengue Virus through Alexa Fluor Labeling

The early events in the interaction between virus and cell can have profound influence on the outcome of infection. Determining the factors 
that influence this interaction could lead to improved understanding of disease pathogenesis and thus influence vaccine or therapeutic design. Hence, the development 
of methods to probe this interaction would be useful. Recent advancements in fluorophores development1-3 and imaging technology4 can be exploited to 
improve our current knowledge on dengue pathogenesis and thus pave the way to reduce the millions of dengue infections occurring annually.

The enveloped dengue virus has an external scaffold consisting of 90 envelope glycoprotein (E) dimers protecting the nucleocapsid shell, 
which contains a single positive strand RNA genome5. The identical protein subunits on the virus surface can thus be labeled with an amine reactive dye and visualized 
through immunofluorescent microscopy. Here, we present a simple method of labeling of dengue virus with Alexa Fluor succinimidyl ester dye dissolved directly in a sodium 
bicarbonate buffer that yielded highly viable virus after labeling. There is no standardized procedure for the labeling of live virus and existing manufacturer&#x2019;s protocol 
for protein labeling usually requires the reconstitution of dye in dimethyl sulfoxide. The presence of dimethyl sulfoxide, even in minute quantities, can block 
productive infection of virus and also induce cell cytotoxicity6. The exclusion of the use of dimethyl sulfoxide in this protocol thus reduced this possibility. 
Alexa Fluor dyes have superior photostability and are less pH-sensitive than the common dyes, such as fluorescein and rhodamine2, making them ideal for 
studies on cellular uptake and endosomal transport of the virus. The conjugation of Alexa Fluor dye did not affect the recognition of labeled dengue 
virus by virus-specific antibody and its putative receptors in host cells7. This method could have useful applications in virological studies.

Taking advantage of the advancements in fluorophore development and imaging technology, a simple method of Alexa Fluor labeling of dengue virus was devised to visualize the early interactions between virus and cell.

The early events in the interaction between virus and cell can have profound influence on the outcome of infection. Determining the factors 
that ...

Use of Artificial Sputum Medium to Test Antibiotic Efficacy Against Pseudomonas aeruginosa in Conditions More Relevant to the Cystic Fibrosis Lung

There is growing concern about the relevance of in vitro antimicrobial susceptibility tests when applied to isolates of P. aeruginosa from cystic fibrosis (CF) patients. Existing methods rely on single or a few isolates grown aerobically and planktonically. Predetermined cut-offs are used to define whether the bacteria are sensitive or resistant to any given antibiotic1. However, during chronic lung infections in CF, P. aeruginosa populations exist in biofilms and there is evidence that the environment is largely microaerophilic2. The stark difference in conditions between bacteria in the lung and those during diagnostic testing has called into question the reliability and even relevance of these tests3.
 Artificial sputum medium (ASM) is a culture medium containing the components of CF patient sputum, including amino acids, mucin and free DNA. P. aeruginosa growth in ASM mimics growth during CF infections, with the formation of self-aggregating biofilm structures and population divergence4,5,6. The aim of this study was to develop a microtitre-plate assay to study antimicrobial susceptibility of P. aeruginosa based on growth in ASM, which is applicable to both microaerophilic and aerobic conditions.
 An ASM assay was developed in a microtitre plate format. P. aeruginosa biofilms were allowed to develop for 3 days prior to incubation with antimicrobial agents at different concentrations for 24 hours. After biofilm disruption, cell viability was measured by staining with resazurin. This assay was used to ascertain the sessile cell minimum inhibitory concentration (SMIC) of tobramycin for 15 different P. aeruginosa isolates under aerobic and microaerophilic conditions and SMIC values were compared to those obtained with standard broth growth. Whilst there was some evidence for increased MIC values for isolates grown in ASM when compared to their planktonic counterparts, the biggest differences were found with bacteria tested in microaerophilic conditions, which showed a much increased resistance up to a &gt;128 fold, towards tobramycin in the ASM system when compared to assays carried out in aerobic conditions.
 The lack of association between current susceptibility testing methods and clinical outcome has questioned the validity of current methods3. Several in vitro models have been used previously to study P. aeruginosa biofilms7, 8. However, these methods rely on surface attached biofilms, whereas the ASM biofilms resemble those observed in the CF lung9 . In addition, reduced oxygen concentration in the mucus has been shown to alter the behavior of P. aeruginosa2 and affect antibiotic susceptibility10. Therefore using ASM under microaerophilic conditions may provide a more realistic environment in which to study antimicrobial susceptibility.

Use of Artificial Sputum Medium to Test Antibiotic Efficacy Against Pseudomonas aeruginosa in Conditions More Relevant to the Cystic Fibrosis Lung

Current diagnostic antimicrobial susceptibility testing relies on the planktonic growth of isolates in nutrient rich, aerobic conditions. Here, we employ an alternative artificial sputum medium to study antimicrobial susceptibility of Pseudomonas aeruginosa biofilms under both aerobic and microaerophilic conditions more representative of the cystic fibrosis lung.

There  is growing concern about the relevance of in vitro antimicrobial  susceptibility tests when applied to isolates of P. aeruginosa from  cystic ...

Culturing Microglia from the Neonatal and Adult Central Nervous System

Microglia are the resident macrophage-like cells of the central nervous system (CNS) and, as such, have critically important roles in physiological and pathological processes such as CNS maturation in development, multiple sclerosis, and spinal cord injury. Microglia can be activated and recruited to action by neuronal injury or stimulation, such as axonal damage seen in MS or ischemic brain trauma resulting from stroke. These immunocompetent members of the CNS are also thought to have roles in synaptic plasticity under non-pathological conditions. We employ protocols for culturing microglia from the neonatal and adult tissues that are aimed to maximize the viable cell numbers while minimizing confounding variables, such as the presence of other CNS cell types and cell culture debris. We utilize large and easily discernable CNS components (e.g. cortex, spinal cord segments), which makes the entire process feasible and reproducible. The use of adult cells is a suitable alternative to the use of neonatal brain microglia, as many pathologies studied mainly affect the postnatal spinal cord. These culture systems are also useful for directly testing the effect of compounds that may either inhibit or promote microglial activation. Since microglial activation can shape the outcomes of disease in the adult CNS, there is a need for in vitro systems in which neonatal and adult microglia can be cultured and studied.

We outline methods for the efficient and quick isolation/culture of viable microglia from the neonatal cerebral cortex and adult spinal cord. The dissection and plating of cortical microglia can be accomplished within 90 minutes, with the subsequent microglial harvest taking place ~ 10 days following the initial dissection.

Microglia are the resident macrophage-like cells of the central nervous system (CNS) and, as such, have critically important roles in physiological and ...

Total Protein Extraction and 2-D Gel Electrophoresis Methods for Burkholderia Species

The investigation of the intracellular protein levels of bacterial species is of importance to understanding the pathogenic mechanisms of diseases caused by these organisms. Here we describe a procedure for protein extraction from Burkholderia species based on mechanical lysis using glass beads in the presence of ethylenediamine tetraacetic acid and phenylmethylsulfonyl fluoride in phosphate buffered saline. This method can be used for different Burkholderia species, for different growth conditions, and it is likely suitable for the use in proteomic studies of other bacteria. Following protein extraction, a two-dimensional (2-D) gel electrophoresis proteomic technique is described to study global changes in the proteomes of these organisms. This method consists of the separation of proteins according to their isoelectric point by isoelectric focusing in the first dimension, followed by separation on the basis of molecular weight by acrylamide gel electrophoresis in the second dimension. Visualization of separated proteins is carried out by silver staining.

Total Protein Extraction and 2-D Gel Electrophoresis Methods for Burkholderia Species

Members of the Burkholderia genus are pathogens of clinical importance. We describe a method for total bacterial protein extraction, using mechanical disruption, and 2-D gel electrophoresis for subsequent proteomic analysis.

The investigation of  the intracellular protein levels of bacterial species is of importance to  understanding the pathogenic mechanisms of diseases ...

Neutrophil Isolation and Analysis to Determine their Role in Lymphoma Cell Sensitivity to Therapeutic Agents

Neutrophils are the most abundant (40% to 75%) type of white blood cells and among the first inflammatory cells to migrate towards the site of inflammation. They are key players in the innate immune system and play major roles in cancer biology. Neutrophils have been proposed as key mediators of malignant transformation, tumor progression, angiogenesis and in the modulation of the antitumor immunity; through their release of soluble factors or their interaction with tumor cells. To characterize the specific functions of neutrophils, a fast and reliable method is coveted for in vitro isolation of neutrophils from human blood. Here, a density gradient separation method is demonstrated to isolate neutrophils as well as mononuclear cells from the blood. The procedure consists of layering the density gradient solution such as Ficoll carefully above the diluted blood obtained from patients diagnosed with chronic lymphocytic leukemia (CLL), followed by centrifugation, isolation of mononuclear layer, separation of neutrophils from RBCsby dextran then lysis of residual erythrocytes. This method has been shown to isolate neutrophils &#8805; 90 % pure. To mimic the tumor microenvironment, 3-dimensional (3D) experiments were performed using basement membrane matrix such as Matrigel. Given the short half-life of neutrophils in vitro, 3D experiments with fresh human neutrophils cannot be performed. For this reason promyelocytic HL60 cells are differentiated along the granulocytic pathway using the differentiation inducers dimethyl sulfoxide (DMSO) and retinoic acid (RA). The aim of our experiments is to study the role of neutrophils on the sensitivity of lymphoma cells to anti-lymphoma agents. However these methods can be generalized to study the interactions of neutrophils or neutrophil-like cells with a large range of cell types in different situations.

Neutrophils are the most abundant type of white blood cells. The isolation of neutrophils from human blood by density gradient separation method and the differentiation of human promyelocytic (HL60) cells along the granulocytic pathway are described here; to test their role on sensitivity of lymphoma cells to anti-lymphoma agents.

Neutrophils are the most abundant (40% to 75%) type of white blood cells and among the first inflammatory cells to migrate towards the site of ...

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. ...

Analysis of Iophenoxic Acid Analogues in Small Indian Mongoose (Herpestes Auropunctatus) Sera for Use as an Oral Rabies Vaccination Biological Marker

The small Indian mongoose (Herpestes auropunctatus) is a reservoir of rabies virus (RABV) in Puerto Rico and comprises over 70% of animal rabies cases reported annually. The control of RABV circulation in wildlife reservoirs is typically accomplished by a strategy of oral rabies vaccination (ORV). Currently no wildlife ORV program exists in Puerto Rico. Research into oral rabies vaccines and various bait types for mongooses has been conducted with promising results. Monitoring the success of ORV relies on estimating bait uptake by target species, which typically involves evaluating a change in RABV neutralizing antibodies (RVNA) post vaccination. This strategy may be difficult to interpret in areas with an active wildlife ORV program or in areas where RABV is enzootic and background levels of RVNA are present in reservoir species. In such situations, a biomarker incorporated with the vaccine or the bait matrix may be useful. We offered 16 captive mongooses placebo ORV baits containing ethyl-iophenoxic acid (et-IPA) in concentrations of 0.4% and 1% inside the bait and 0.14% in the external bait matrix. We also offered 12 captive mongooses ORV baits containing methyl-iophenoxic acid (me-IPA) in concentrations of 0.035%, 0.07% and 0.14% in the external bait matrix. We collected a serum sample prior to bait offering and then weekly for up to eight weeks post offering. We extracted Iophenoxic acids from sera into acetonitrile and quantified using liquid chromatography/mass spectrometry. We analyzed sera for et-IPA or me-IPA by liquid chromatography-mass spectrometry. We found adequate marking ability for at least eight and four weeks for et- and me-IPA, respectively. Both IPA derivatives could be suitable for field evaluation of ORV bait uptake in mongooses. Due to the longevity of the marker in mongoose sera, care must be taken to not confound results by using the same IPA derivative during consecutive evaluations.

Analysis of Iophenoxic Acid Analogues in Small Indian Mongoose Herpestes Auropunctatus Sera for Use as an Oral Rabies Vaccination Biological Marker

We offered captive mongooses placebo oral rabies vaccine baits with ethyl or methyl iophenoxic acid as a biomarker and verified bait uptake using a novel liquid chromatography with tandem mass spectrometry (LC-MS/MS) method.

The small Indian mongoose (Herpestes auropunctatus) is a reservoir of rabies virus (RABV) in Puerto Rico and comprises over 70% of animal rabies cases ...

Recurrent Escherichia coli Urinary Tract Infection Triggered by Gardnerella vaginalis Bladder Exposure in Mice

Recurrent urinary tract infections (rUTI) caused by uropathogenic Escherichia coli (UPEC) are common and costly. Previous articles describing models of UTI in male and female mice have illustrated the procedures for bacterial inoculation and enumeration in urine and tissues. During an initial bladder infection in C57BL/6 mice, UPEC establish latent reservoirs inside bladder epithelial cells that persist following clearance of UPEC bacteriuria. This model builds on these studies to examine rUTI caused by the emergence of UPEC from within latent bladder reservoirs. The urogenital bacterium Gardnerella vaginalis is used as the trigger of rUTI in this model because it is frequently present in the urogenital tracts of women, especially in the context of vaginal dysbiosis that has been associated with UTI. In addition, a method for in situ bladder fixation followed by scanning electron microscopy (SEM) analysis of bladder tissue is also described, with potential application to other studies involving the bladder.

Recurrent Escherichia coli Urinary Tract Infection Triggered by Gardnerella vaginalis Bladder Exposure in Mice

A mouse model of uropathogenic E. coli (UPEC) transurethral inoculation to establish latent intracellular bladder reservoirs and subsequent bladder exposure to G. vaginalis to induce recurrent UPEC UTI is demonstrated. Also demonstrated are the enumeration of bacteria, urine cytology, and in situ bladder fixation and processing for scanning electron microscopy.

Recurrent urinary tract infections (rUTI) caused by uropathogenic Escherichia coli (UPEC) are common and costly. Previous articles describing models of ...

Separation of Immune Cell Subpopulations in Peripheral Blood Samples from Children with Infectious Mononucleosis

Infectious mononucleosis (IM) is an acute syndrome mostly associated with primary Epstein&#8211;Barr virus (EBV) infection. The main clinical symptoms include irregular fever, lymphadenopathy, and significantly increased lymphocytes in peripheral blood. The pathogenic mechanism of IM is still unclear; there is no effective treatment method for it, with mainly symptomatic therapies being available. The main question in EBV immunobiology is why only a small subset of infected individuals shows severe clinical symptoms and even develop EBV-associated malignancies, whilemost individuals are asymptomatic for life with the virus.
B cells are first involved in IM because EBV receptors are presented on their surface. Natural killer (NK) cells are cytotoxic innate lymphocytes that are important for killing EBV-infected cells. The proportion of CD4+ T cells decreases while that of CD8+ T cells expands dramatically during acute EBV infection, and the persistence of CD8+ T cells is important for lifelong control of IM. Those immune cells play important roles in IM, and their functions need to be identified separately. For this purpose, monocytes are separated first from peripheral blood mononuclear cells (PBMCs) of IM individuals using CD14 microbeads, a column, and a magnetic separator.
The remaining PBMCs are stained with peridinin-chlorophyll-protein (PerCP)/Cyanine 5.5 anti-CD3, allophycocyanin (APC)/Cyanine 7 anti-CD4, phycoerythrin (PE) anti-CD8, fluorescein isothiocyanate (FITC) anti-CD19, APC anti-CD56, and APC anti-CD16 antibodies to sort CD4+ T cells, CD8+ T cells, B cells, and NK cells using a flow cytometer. Furthermore, transcriptome sequencing of five subpopulations was performed to explore their functions and pathogenic mechanisms in IM.

We describe a method combining immunomagnetic beads and fluorescence-activated cell sorting to isolate and analyze defined immune cell subpopulations of peripheral blood mononuclear cells (monocytes, CD4+ T cells, CD8+ T cells, B cells, and natural killer cells). Using this method, magnetic and fluorescently labeled cells can be purified and analyzed.

Infectious mononucleosis (IM) is an acute syndrome mostly associated with primary Epstein&#8211;Barr virus (EBV) infection. The main clinical symptoms ...

Standardization of Transfer across Labs between Flow Cytometers for Detection of Lymphocytes in Japanese Encephalitis Vaccinated Children

An increasing number of laboratories need to collect data from multiple flow cytometers, especially for research projects performed across multiple centers. The challenges of using two flow cytometers in different labs include the lack of standardized materials, software compatibility issues, inconsistencies in instrument setup, and the use of different configurations for different flow cytometers. To establish a standardized flow cytometry experiment to achieve the consistency and comparability of experimental results across multiple centers, a rapid and feasible standardization method was established to transfer parameters across different flow cytometers. 
The methods developed in this study allowed the transfer of experimental settings and analysis templates between two flow cytometers in different laboratories for the detection of lymphocytes in Japanese encephalitis (JE)-vaccinated children. A consistent fluorescence intensity was obtained between the two cytometers using fluorescence standard beads to establish the cytometer settings. Comparable results were obtained in two laboratories with different types of instruments. Using this method, we can standardize analysis for evaluating the immune function of JE-vaccinated children in different laboratories with different instruments, diminish the differences in data and results among flow cytometers in multiple centers, and provide a feasible approach for the mutual accreditation of laboratory results. The standardization method of flow cytometer experiments will ensure the effective performance of research projects across multiple centers.

In this study, a method was developed to facilitate the transfer of experimental settings and analysis templates between two flow cytometers in two laboratories for the detection of lymphocytes in Japanese encephalitis-vaccinated children. The standardization method for the flow cytometer experiments will allow research projects to be effectively conducted in multiple centers.

An increasing number of laboratories need to collect data from multiple flow cytometers, especially for research projects performed across multiple ...