Name: protocols for investigating the host-tissue distribution, transmission-mode, and effect on the host fitness of a densovirus in the cotton bollworm
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Description: Watch this Scientific Journal Video about Protocols for Investigating the Host-tissue Distribution, Transmission-mode, and Effect on the Host Fitness of a Densovirus in the Cotton Bollworm at JoVE.com

This work was supported by the National Key Basic Research Program of China (No. 2013CB127602) and the Science Fund for Creative Research Groups of the National Science Foundation of China (No. 31321004).

1. Construction of the HaDV2-free Cotton Bollworm Colony
<ol>
	<li>Rear cotton bollworm larvae (H. armigera) on an artificial diet32 in a controlled growth room or an artificial climate chamber at 25 &#177; 1 &#176;C, with 14 h light/10 h dark and 60% relative humidity.</li>
	<li>Aid single-pair mating of newly eclosed moths by using a plastic cage per pair (10 cm height, 5 cm diameter) covered with cotton gauze to ensure good ventilation and to serve as an oviposition substrate. To inc...

<ol>
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S., 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=Herpesvirus+latency+confers+symbiotic+protection+from+bacterial+infection.">Herpesvirus latency confers symbiotic protection from bacterial infection.</a> Nature. 447, 326-329 (2007).</li><li>Ponferrada, V. G., Mauck, B. S., Wooley, D. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+envelope+glycoprotein+of+human+endogenous+retrovirus+herv-w+induces+cellular+resistance+to+spleen+necrosis+virus.">The envelope glycoprotein of human endogenous retrovirus herv-w induces cellular resistance to spleen necrosis virus.</a> Arch. Virol. 148, 659-675 (2003).</li><li>M&#225;rquez, L. M., Redman, R. S., Rodriguez, R. J., Roossinck, M. 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+virus+in+a+fungus+in+a+plant:+three-way+symbiosis+required+for+thermal+tolerance.">A virus in a fungus in a plant: three-way symbiosis required for thermal tolerance.</a> Science. 315, 513-515 (2007).</li><li>Xu, P. 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=Complete+genome+sequence+of+a+monosense+densovirus+infecting+the+cotton+bollworm,+Helicoverpa+armigera.">Complete genome sequence of a monosense densovirus infecting the cotton bollworm, Helicoverpa armigera.</a> J. Virol. 86, 10909-10909 (2012).</li><li>Xu, P. J., Liu, Y. Q., Graham, R. I., Wilson, K., Wu, K. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Densovirus+is+a+mutualistic+symbiont+of+a+global+crop+pest+(Helicoverpa+armigera)+and+protects+against+a+baculovirus+and+Bt+biopesticide.">Densovirus is a mutualistic symbiont of a global crop pest (Helicoverpa armigera) and protects against a baculovirus and Bt biopesticide.</a> PLoS Pathog. 10, e1004490 (2014).</li><li>Liang, G. M., Tan, W. J., Guo, Y. Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+improvement+in+the+technique+of+arti&#64257;cial+rearing+cotton+bollworm.">An improvement in the technique of arti&#64257;cial rearing cotton bollworm.</a> Plant Protec. 25, 15-17 (1999).</li><li>Zhou, W. 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In the last few decades, most studies on insect-virus interactions have focused on honey bee health34,35,36, vectors of human disease37, plant viruses38, and some insect pathogenic viruses that have great potential as biological control agents39. Little attention has been paid to covert viruses in insects, especially in lepidopteran pests. Here, we present a...

In the last few decades, the development of sequencing technology such as next-generation sequencing (NGS) has facilitated the discovery of many novel DNA and RNA viruses, especially nonpathogenic viruses, but also novel isolates of previously known viruses1,2,3,4,5,6,7,8,9,10,11,12. In the model organism Drosophila melanogaster, more than 20 new partial virus genomes have been detected using metagenomic techniques13. Many viral sequences, including novel viruses, have also been identified in other insects, such as honey bees, mosquitoes, Asian citrus psyllids, dragonflies, and multiple lepidopteran species14,15,16,17,18,19,20,21.
In the future, it can be expected that more novel viruses will be discovered in insects using these advanced technologies; hence, our understanding of the virus-host interaction may change accordingly6,9. For example, virus-host interactions are considered to be more complicated than previously thought, because many novel viruses are being defined as mutualistic partners rather than strict pathogens22. For example, the mutualistic densovirus DplDNV in Dysaphis plantaginea induces the winged morph and increases mobility, facilitating the dispersal of the host as well as the virus23. Moreover, mutualistic viruses have been described with regards to mammalian health, the drought and cold tolerance of plants, and the impact of bacterial infections24. Seneca valley virus-001 is shown to mediate selective cytotoxicity towards tumor cells with neuroendocrine cancer features25. Hepatitis A virus infections suppress hepatitis C virus replication and may lead to recovery from hepatitis C26. Herpesvirus latency confers symbiotic protection from bacterial infection27. The envelope glycoprotein of human endogenous retrovirus HERV-W induces cellular resistance to spleen necrosis virus28. Curvularia thermal tolerance virus (CThTV) from a fungal endophyte is involved in the mutualistic interaction between this fungus and a tropical panic grass29. Hence, knowledge of the interactions between the newly found viruses and their hosts should generate fresh perspectives on their biology and management. However, the novel viruses, especially the covert viruses displaying no obvious signs typical of acute infection, have seldom been investigated, and we need a pipeline and protocols to investigate the impacts of the newly found viruses on their hosts.
Previously, we have reported the prevalence a new monosense densovirus Helicoverpa armigera densovirus (HaDV2) in the cotton bollworm, Helicoverpa armigera, and presented evidence of a mutualistic relationship between HaDV2 and the cotton bollworm30,31. In this paper, we will describe the laboratory protocol to study in detail the interaction between HaDV2 and its cotton bollworm host. The protocol presented here may also be highly relevant to researchers examining the role of other orally-transmitted viruses, especially in lepidopteran pests.

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			<td>24-well plate</td>
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			<td>07-200-740</td>
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			<td>DNA extraction kit</td>
			<td>TIANGEN</td>
			<td>DP304-03</td>
			<td>Multiple suppliers available.</td>
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			<td>thermal cycler</td>
			<td>Veriti; Applied Biosystems</td>
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			<td>pEASY-T Cloning Vector</td>
			<td>TransGen, Beijing, China</td>
			<td>CT301-02</td>
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			<td>IDEAL-TEK</td>
			<td>2.SA</td>
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			<td>Premix Ex Taq (Probe qPCR)</td>
			<td>Takara</td>
			<td>RR390A</td>
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			<td>Probes</td>
			<td>Invitrogen</td>
			<td>Custom order</td>
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			<td>Primers</td>
			<td>Invitrogen</td>
			<td>Custom order</td>
			<td></td>
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			<td>microspectrophotometry NanoDrop 2000c&#160;</td>
			<td>Thermo scientific&#160;</td>
			<td>not available</td>
			<td></td>
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			<td>7500 Real-Time PCR system</td>
			<td>Applied Biosystems</td>
			<td>not available</td>
			<td></td>
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			<td>stereomicroscope SZX-16</td>
			<td>Olympus</td>
			<td>not available</td>
			<td></td>
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			<td>sucrose</td>
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			<td></td>
			<td>Multiple suppliers available.</td>
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			<td>vitamin complex</td>
			<td></td>
			<td></td>
			<td>Multiple suppliers available.</td>
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protocols for investigating the host-tissue distribution, transmission-mode, and effect on the host fitness of a densovirus in the cotton bollworm

Many novel viruses have been discovered in animal hosts using next-generation sequencing technologies. Previously, we reported a mutualistic virus, Helicoverpa armigera densovirus (HaDV2), in a lepidopteran species, the cotton bollworm, Helicoverpa armigera (Hubner). Here, we describe the protocols that are currently used to study the effect of HaDV2 on its host. First, we establish a HaDV2-free cotton bollworm colony from a single breeding pair. Then, we orally inoculate some neonate larval offspring with HaDV2-containing filtered liquid to produce two colonies with the same genetic background: one HaDV2-infected, the other uninfected. A protocol to compare life table parameters (e.g., larval, pupal, and adult periods and fecundity) between the HaDV2-infected and -uninfected individuals is also presented, as are the protocols for determining the host-tissue distribution and transmission efficiency of HaDV2. These protocols would also be suitable for investigating the effects of other orally transmitted viruses on their insect hosts, lepidopteran hosts in particular.

Progenies from the parents that were HaDV2-free (Figure 3A) were reared as the NONINF-strain. We successfully amplified the actin gene using the same DNA templates, suggesting that the DNA templates were of good quality (Figure 3B). In addition, the randomly selected eight progenies were also free of HaDV2 (Figure 3C), HaNPV (Figure 3D), and Wolbachia (Figure 3E). Again, we concluded tha...

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Protocols for Investigating the Host-tissue Distribution, Transmission-mode, and Effect on the Host Fitness of a Densovirus in the Cotton Bollworm

Here, we present a protocol to investigate the host-tissue distribution, transmission mode, and effect on the host fitness of a densovirus within a lepidopteran species, the cotton bollworm. This protocol can also be used for studying the interaction between other orally-transmitted viruses and their insect hosts.

Many novel viruses have been discovered in animal hosts using next-generation sequencing technologies. Previously, we reported a mutualistic virus, ...

The overall goal of this protocol is to study the effect of a Densovirus, HaDV2, on its host, Cotton Bollworm. This method can help answer key questions in the virus host interaction field such as the effect a now path-standing viruses on their hosts. The main advantage of this technique that larvae use to construct virus positive and negative strains are derived from the same par-ne-co-hosts, ensuring the same genetic background.To begin the experiment, eight single-pair mating of newly enclosed moths by using a plastic cage per pair covered with cotton gauze to ensure good ventilation and to serve as an ovum position substrate. To increase the likelihood of obtaining a virus-free colony, utilize at least 30 pairs of single-pair matings. Feed adult moths with a 10%sugar and 2%vitamin solution-soaked cotton ball, and replenish the solution daily.Today's post-mating, as female adults lay their eggs on the cotton gauze, begin to change the gauze daily. Collect and maintain gauze-containing eggs in the same growth room as the adult moths until the eggs hatch. Immediately transfer newly hatched larvae to new 24-well plates with one individual per well.Five days after the first egg-laying date, collect the parent moths and store them in liquid nitrogen. Check for HaDV2 of the parents using PCR with specific primers, and keep the HaDV2-free individuals as the HaDV2-free noninfected strain. Collect adult moths individually and immediately store them in liquid nitrogen.Completely grind collected individual moths in liquid nitrogen using a sterilized mortar and pestle. Transfer around 10 micrograms of debris into a clean 1.5 milliliter tube. Then transfer the remaining debris into a new tube then immediately store them at 80 degrees Celsius.Extract DNA from the 10 micrograms of debris following the protocols provided by the manufacturer. Once the moth DNA has been isolated, check for HaDV2 using the protocol. According to the results, divide the remaining debris into two groups:HaDV2 positive and HaDV2 negative.Add one milliliter of phosphate-buffered saline, or PBS, to each individual to completely dissolve the remaining larval debris. Pre-cool the centrifuge four degrees Celsius, then spin the homogenate. On ice, filter the liquid supernatant with the 0.22 micron membrane filter.Collect 200 microliters per tube of the filtered liquids, and immediately store them at 80 degrees Celsius. To determine the oral infection efficiency with the HaDV2 infected filtered liquids of different concentration by diluting the fluid containing HaDV2. In a fume hood evenly spread 200 microliters of HaDV2 containing filtered liquids for each concentration on the surface of the solid artifical diet.Next, begin to transfer 100 freshly hatched larvae to the HaDV2 containing artificial diet in a petri dish. Gently knock the cotton gauze including freshly hatched larvae to transfer the larvae from the gauze to a sheet of white paper. Knock the paper gently so that the larvae spin silk and balloon off the paper.Use sterilized forceps to touch the silk and move the larvae to a petri dish. After 48 hours, transfer one individual larvae per well on the artificial diet to 24-well plates. Re-ar the larvae inoculated with HaDV2, using the same concentrations to the adult stage.Check for HaDV2 infection status of the adults using the protocol. Adults inoculated with 10 to the eighth copy number per microliter of HaDV2 yielded 100%infection and are kept as HaDV2 positive strain. To prevent contamination, sterilize the forceps and scissors in 75%ethanol three times and then heat them in the flame of an alcohol lamp.Using an analytical balance weigh the empty tubes that are to be used to store the tissues. Next place the larvae and adult moths from the infected strain on ice for approximately five minutes. Fix the frozen larvae on cystosepiment using two insect pins inserted in the intersegmental membranes near the head and the last abdomen segment.Then in the posterior abdomen, cut the proleg of the larvae so that the hemolymph flows out. Use a pipette to pipette about 10 microliters of larval hemolymph within about 10 seconds to prevent browning and coagulating of the hemolymph. With a pair of scissors make an incision in the cuticle ranging from the last intersegmental membrane to the head.Using two forceps, dissect foregut, midgut, hindgut, fat body, and malpighian tubules. To prevent the contamination of the remaining tissues with the hemolymph during the dissection, wash the dissected tissues in PBS buffer. Mix tissue samples from three individuals together as one replicant.Immediately transfer each tissue to the tubes in liquid nitrogen. In a clean petri dish containing PBS buffer, dissect the tissues of female and male adults and immediately transfer each tissue to the tubes in liquid nitrogen. Weigh the tubes and the above tissues together separately and calculate the mass of tissues.Extract DNA from these tissues and perform kPCR to quantify the viral titer of each tissue. Place at least 100 pupae or newly emerged adult moths derived from the noninfected strain in a cotton gauze-covered 5-liter wire mesh cage. After two days, mated adult females will start to lay eggs on the cotton gauze.Collect and maintain the cotton gauze containing the eggs. Proceed with neonate larvae inoculation by transferring the neonate larvae of the noninfected strain to the artificial diet containing HaDV2 to establish the infected strain. Similarly, transfer neonate larvae to the artificial diet covered with HaDV2-free filtered liquid to establish the control groups, the noninfected strain.Include at least 240 larvae in each treatment. After 48 hours transfer one individual larva per well to 24-well plates. Sequentially number each larva in each well, and record the instar stage and survival status daily.About five days later, when the artificial diet deteriorates, simultaneously transfer these individuals to a new 24-well plate containing fresh artificial diet. Weigh the larvae from day seven to 11 using an analytical balance. After weighing, individually return the larvae to the 24-well plate.Upon molting to the fifth instar, around ten day after hatching, transfer the larvae to glass tubes. Transfer the individuals to the glass tube at the same time each day. Fifth instar larvae can also be identified by the width of the head capsule.Next, code each larva with its original reference number on the glass tube using paint or markers. Record the larval status daily. Record the pupae and eclosion date for each individual in the glass tube, and record the mass of three-day old pupae.After eclosion, place one female and one male into a single cage with 10%sucrose and 2%vitamin solution. Replenish the sucrose solution every day. Record the date of death for each moth.When eggs are laid, change the cotton gauze daily. Count the number of eggs immediately. Finally, count newly hatched offspring while they hatch.Tri-als were run to ensure the quality of the specimens used in the experiment. Progenies from the male and female parents that we HaDV2-free were reared as the noninfected strain. We successfully amplified the actin gene in parents and progenies using the same DNA template suggesting that the DNA templates were of good quality.The randomly selected eight progenies were also free of HaDV2, HaNPV, and Wolbachia. A standard curve defining the relationship between the log of the quality of starting material and the gained CT values shows a strong linear correlation between the CT values and the log of the initial gene copy number of HaDV2. The HaDV2 copy number was calculated per microgram of each tissue, and the percentage of the total HaDV2 titer for the specific tissue revealed that HaDV2 was mainly distributed in the fat body.While attempting this procedure, it is important to remember to handle the delicate larvae during the first five days after they hatch during the en-tire procedure.

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In vivo Imaging of Transgenic Leishmania Parasites in a Live Host

Distinct species of Leishmania, a protozoan parasite of the family Trypanosomatidae, typically cause different human disease manifestations. The most common forms of disease are visceral leishmaniasis (VL) and cutaneous leishmaniasis (CL). Mouse models of leishmaniasis are widely used, but quantification of parasite burdens during murine disease requires mice to be euthanized at various times after infection. Parasite loads are then measured either by microscopy, limiting dilution assay, or qPCR amplification of parasite DNA. The in vivo imaging system (IVIS) has an integrated software package that allows the detection of a bioluminescent signal associated with cells in living organisms. Both to minimize animal usage and to follow infection longitudinally in individuals, in vivo models for imaging Leishmania spp. causing VL or CL were established. Parasites were engineered to express luciferase, and these were introduced into mice either intradermally or intravenously. Quantitative measurements of the luciferase driving bioluminescence of the transgenic Leishmania parasites within the mouse were made using IVIS. Individual mice can be imaged multiple times during longitudinal studies, allowing us to assess the inter-animal variation in the initial experimental parasite inocula, and to assess the multiplication of parasites in mouse tissues. Parasites are detected with high sensitivity in cutaneous locations. Although it is very likely that the signal (photons/second/parasite) is lower in deeper visceral organs than the skin, but quantitative comparisons of signals in superficial versus deep sites have not been done. It is possible that parasite numbers between body sites cannot be directly compared, although parasite loads in the same tissues can be compared between mice. Examples of one visceralizing species (L. infantum chagasi) and one species causing cutaneous leishmaniasis (L. mexicana) are shown. The IVIS procedure can be used for monitoring and analyzing small animal models of a wide variety of Leishmania species causing the different forms of human leishmaniasis.

In vivo Imaging of Transgenic Leishmania Parasites in a Live Host

An in vivo imaging system is used to generate quantitative measurements of murine infection with the Trypanosomatid protozoan Leishmania. This is a non-invasive and non-lethal method for detecting parasites expressing luciferase within many tissues throughout the course of chronic Leishmania spp. infection.

Distinct species of Leishmania, a protozoan parasite of the family Trypanosomatidae, typically cause different human disease manifestations. The most ...

Use of the EpiAirway Model for Characterizing Long-term Host-pathogen Interactions

Nontypeable Haemophilus influenzae (NTHi) are human-adapted Gram-negative bacteria that can cause recurrent and chronic infections of the respiratory mucosa 1; 2. To study the mechanisms by which these organisms survive on and inside respiratory tissues, a model in which successful long-term co-culture of bacteria and human cells can be performed is required. We use primary human respiratory epithelial tissues raised to the air-liquid interface, the EpiAirway model (MatTek, Ashland, MA). These are non-immortalized, well-differentiated, 3-dimensional tissues that contain tight junctions, ciliated and nonciliated cells, goblet cells that produce mucin, and retain the ability to produce cytokines in response to infection. 
This biologically relevant in vitro model of the human upper airway can be used in a number of ways; the overall goal of this method is to perform long-term co-culture of EpiAirway tissues with NTHi and quantitate cell-associated and internalized bacteria over time. As well, mucin production and the cytokine profile of the infected co-cultures can be determined. This approach improves upon existing methods in that many current protocols use submerged monolayer or Transwell cultures of human cells, which are not capable of supporting bacterial infections over extended periods3. For example, if an organism can replicate in the overlying media, this can result in unacceptable levels of cytotoxicity and loss of host cells, arresting the experiment. The EpiAirway model allows characterization of long-term host-pathogen interactions. Further, since the source for the EpiAirway is normal human tracheo-bronchial cells rather than an immortalized line, each is an excellent representation of actual human upper respiratory tract tissue, both in structure and in function4.
For this method, the EpiAirway tissues are weaned off of anti-microbial and anti-fungal compounds for 2 days prior to delivery, and all procedures are performed under antibiotic-free conditions. This necessitates special considerations, since both bacteria and primary human tissues are used in the same biosafety cabinet, and are co-cultured for extended periods.

This method allows characterization of extended bacterial co-culture with EpiAirways, primary human respiratory epithelial tissue grown at the air-liquid interface, a biologically relevant in vitro model. The approach can be used with any microbe that is amenable to long-term co-culture.

Nontypeable Haemophilus influenzae  (NTHi) are human-adapted Gram-negative bacteria that can cause recurrent and chronic infections of the respiratory ...

Induction of Graft-versus-host Disease and In Vivo T Cell Monitoring Using an MHC-matched Murine Model

Graft-versus-host disease (GVHD) is the limiting barrier to the broad use of bone marrow transplant as a curative therapy for a variety of hematological deficiencies. GVHD is caused by mature alloreactive T cells present in the bone marrow graft that are infused into the recipient and cause damage to host organs. However, in mice, T cells must be added to the bone marrow inoculum to cause GVHD. Although extensive work has been done to characterize T cell responses post transplant, bioluminescent imaging technology is a non-invasive method to monitor T cell trafficking patterns in vivo. 
Following lethal irradiation, recipient mice are transplanted with bone marrow cells and splenocytes from donor mice. T cell subsets from L2G85.B6 (transgenic mice that constitutively express luciferase) are included in the transplant. By only transplanting certain T cell subsets, one is able to track specific T cell subsets in vivo, and based on their location, develop hypotheses regarding the role of specific T cell subsets in promoting GVHD at various time points. At predetermined intervals post transplant, recipient mice are imaged using a Xenogen IVIS CCD camera. Light intensity can be quantified using Living Image software to generate a pseudo-color image based on photon intensity (red = high intensity, violet = low intensity). 
Between 4-7 days post transplant, recipient mice begin to show clinical signs of GVHD. Cooke et al.1 developed a scoring system to quantitate disease progression based on the recipient mice fur texture, skin integrity, activity, weight loss, and posture. Mice are scored daily, and euthanized when they become moribund. Recipient mice generally become moribund 20-30 days post transplant. 
Murine models are valuable tools for studying the immunology of GVHD. Selectively transplanting particular T cell subsets allows for careful identification of the roles each subset plays. Non-invasively tracking T cell responses in vivo adds another layer of value to murine GVHD models.

Induction of Graft-versus-host Disease and In Vivo T Cell Monitoring Using an MHC-matched Murine Model

Murine bone marrow transplantation is a widely used technique to study immunological mechanisms governing graft-versus-host disease in humans. The ability to monitor T cell trafficking patterns in vivo allows for detailed analysis of the development and perpetuation of T cell responses during graft-versus-host disease.

Graft-versus-host disease (GVHD) is the limiting barrier to the broad use of bone marrow transplant as a curative therapy for a variety of hematological ...

The Synergistic Effect of Visible Light and Gentamycin on Pseudomona aeruginosa Microorganisms

Recently there were several publications on the bactericidal effect of visible light, most of them claiming that blue part of the spectrum (400 nm-500 nm) is responsible for killing various pathogens1-5. The phototoxic effect of blue light was suggested to be a result of light-induced reactive oxygen species (ROS) formation by endogenous bacterial photosensitizers which mostly absorb light in the blue region4,6,7. There are also reports of biocidal effect of red and near infra red8 as well as green light9.
In the present study, we developed a method that allowed us to characterize the effect of high power green (wavelength of 532 nm) continuous (CW) and pulsed Q-switched (Q-S) light on Pseudomonas aeruginosa. Using this method we also studied the effect of green light combined with antibiotic treatment (gentamycin) on the bacteria viability. P. aeruginosa is a common noscomial opportunistic pathogen causing various diseases. The strain is fairly resistant to various antibiotics and contains many predicted AcrB/Mex-type RND multidrug efflux systems10.
The method utilized free-living stationary phase Gram-negative bacteria (P. aeruginosa strain PAO1), grown in Luria Broth (LB) medium exposed to Q-switched and/or CW lasers with and without the addition of the antibiotic gentamycin. Cell viability was determined at different time points. The obtained results showed that laser treatment alone did not reduce cell viability compared to untreated control and that gentamycin treatment alone only resulted in a 0.5 log reduction in the viable count for P. aeruginosa. The combined laser and gentamycin treatment, however, resulted in a synergistic effect and the viability of P. aeruginosa was reduced by 8 log's.
The proposed method can further be implemented via the development of catheter like device capable of injecting an antibiotic solution into the infected organ while simultaneously illuminating the area with light.

The Synergistic Effect of Visible Light and Gentamycin on Pseudomona aeruginosa Microorganisms

We show that a developed biomedical device involving continuous or pulsed visible laser based treatment that is combined with antibiotic treatment (gentamycin), results in a statistically significant synergistic effect leading to a reduction in the viability of P. aeruginosa PAO1, by 8 log's compared to antibiotic treatment alone.

Recently there were several publications on the  bactericidal effect of visible light, most of them claiming that blue part of  the spectrum (400 nm-500 ...

Using Click Chemistry to Measure the Effect of Viral Infection on Host-Cell RNA Synthesis

Many RNA viruses have evolved the ability to inhibit host cell transcription as a means to circumvent cellular defenses. For the study of these viruses, it is therefore important to have a quick and reliable way of measuring transcriptional activity in infected cells. Traditionally, transcription has been measured either by incorporation of radioactive nucleosides such as 3H-uridine followed by detection via autoradiography or scintillation counting, or incorporation of halogenated uridine analogs such as 5-bromouridine (BrU) followed by detection via immunostaining. The use of radioactive isotopes, however, requires specialized equipment and is not feasible in a number of laboratory settings, while the detection of BrU can be cumbersome and may suffer from low sensitivity.
The recently developed click chemistry, which involves a copper-catalyzed triazole formation from an azide and an alkyne, now provides a rapid and highly sensitive alternative to these two methods. Click chemistry is a two step process in which nascent RNA is first labeled by incorporation of the uridine analog 5-ethynyluridine (EU), followed by detection of the label with a fluorescent azide. These azides are available as several different fluorophores, allowing for a wide range of options for visualization.
This protocol describes a method to measure transcriptional suppression in cells infected with the Rift Valley fever virus (RVFV) strain MP-12 using click chemistry. Concurrently, expression of viral proteins in these cells is determined by classical intracellular immunostaining. Steps 1 through 4 detail a method to visualize transcriptional suppression via fluorescence microscopy, while steps 5 through 8 detail a method to quantify transcriptional suppression via flow cytometry. This protocol is easily adaptable for use with other viruses.

This method describes the use of click chemistry to measure changes in host cell transcription after infection with the Rift Valley fever virus (RVFV) strain MP-12. Results can be visualized qualitatively via fluorescence microscopy or obtained quantitatively through flow cytometry. This method is adaptable for use with other viruses.

Many RNA viruses have evolved the ability to inhibit host cell transcription as a means to circumvent cellular defenses. For the study of these viruses, ...

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 ...

Bioenergetics and the Oxidative Burst: Protocols for the Isolation and Evaluation of Human Leukocytes and Platelets

Mitochondrial dysfunction is known to play a significant role in a number of pathological conditions such as atherosclerosis, diabetes, septic shock, and neurodegenerative diseases but assessing changes in bioenergetic function in patients is challenging.&#160;Although diseases such as diabetes or atherosclerosis present clinically with specific organ impairment, the systemic components of the pathology, such as hyperglycemia or inflammation, can alter bioenergetic function in circulating leukocytes or platelets. This concept has been recognized for some time but its widespread application has been constrained by the large number of primary cells needed for bioenergetic analysis.&#160;This technical limitation has been overcome by combining the specificity of the magnetic bead isolation techniques, cell adhesion techniques, which allow cells to be attached without activation to microplates, and the sensitivity of new technologies designed for high throughput microplate respirometry.&#160;An example of this equipment is the extracellular flux analyzer. Such instrumentation typically uses oxygen and pH sensitive probes to measure rates of change in these parameters in adherent cells, which can then be related to metabolism. Here we detail the methods for the isolation and plating of monocytes, lymphocytes, neutrophils and platelets, without activation, from human blood and the analysis of mitochondrial bioenergetic function in these cells. In addition, we demonstrate how the oxidative burst in monocytes and neutrophils can also be measured in the same samples. Since these methods use only 8-20 ml human blood they have potential for monitoring reactive oxygen species generation and bioenergetics in a clinical setting.

Blood leukocytes and platelets can be used as a marker of overall bioenergetic health of an individual and so have the potential to monitor pathological processes and the impact of treatments. Here&#160;we describe a method to isolate and measure mitochondrial function and the oxidative burst in these cells.

Mitochondrial dysfunction is known to play a significant role in a number of pathological conditions such as atherosclerosis, diabetes, septic shock, and ...

Human Placental and Decidual Organ Cultures to Study Infections at the Maternal-fetal Interface

The placenta shows a large degree of interspecies anatomic variability. To best understand biology and pathophysiology of the human placenta, it is imperative to design experiments using human cells and tissues. An advantage of organ culture is maintenance of three-dimensional (3D) structural organization and extracellular matrix. The goal of the method described here is successful establishment of ex vivo human gestational tissue organ cultures and their healthy culture maintenance for 72-96 hr. The protocol details the immediate processing of research-consented, placental and decidual specimens fresh from the operating suite. These are abundant specimens that would otherwise be discarded. Detailed instructions on the sterile collection of these samples, including morphologic details on how to select appropriate tissues to establish 3D organ cultures, is provided. Placental villous and decidual tissues are microdissected into 2-3 mm3 pieces and placed separately on matrix-lined transwell filters and cultured for several days. Villous and decidual organ cultures are well suited for the study of human host-pathogen interaction. As compared to other model organisms, these human cultures are particularly advantageous to examine mechanism of infection for pathogens that demonstrate variable patterns of host specificity. As an example, we demonstrate infection of placental and decidual organ cultures with the clinically relevant, facultative intracellular bacterial pathogen Listeria monocytogenes.

A simple method to establish primary human placental (villous) and decidual organ cultures is described. Villous and decidual organ cultures are invaluable tools for studying pathogenesis at the human maternal-fetal interface. Infection with the facultative intracellular bacterium Listeria monocytogenes is demonstrated.

The placenta shows a large degree of interspecies anatomic variability. To best understand biology and pathophysiology of the human placenta, it is ...

Implementation of a Permeable Membrane Insert-based Infection System to Study the Effects of Secreted Bacterial Toxins on Mammalian Host Cells

Many bacterial pathogens secrete potent toxins to aid in the destruction of host tissue, to initiate signaling changes in host cells or to manipulate immune system responses during the course of infection. Though methods have been developed to successfully purify and produce many of these important virulence factors, there are still many bacterial toxins whose unique structure or extensive post-translational modifications make them difficult to purify and study in in vitro systems. Furthermore, even when pure toxin can be obtained, there are many challenges associated with studying the specific effects of a toxin under relevant physiological conditions. Most in vitro cell culture models designed to assess the effects of secreted bacterial toxins on host cells involve incubating host cells with a one-time dose of toxin. Such methods poorly approximate what host cells actually experience during an infection, where toxin is continually produced by bacterial cells and allowed to accumulate gradually during the course of infection. This protocol describes the design of a permeable membrane insert-based bacterial infection system to study the effects of Streptolysin S, a potent toxin produced by Group A Streptococcus, on human epithelial keratinocytes. This system more closely mimics the natural physiological environment during an infection than methods where pure toxin or bacterial supernatants are directly applied to host cells. Importantly, this method also eliminates the bias of host responses that are due to direct contact between the bacteria and host cells. This system has been utilized to effectively assess the effects of Streptolysin S (SLS) on host membrane integrity, cellular viability, and cellular signaling responses. This technique can be readily applied to the study of other secreted virulence factors on a variety of mammalian host cell types to investigate the specific role of a secreted bacterial factor during the course of infection.

Here, a method using a permeable membrane insert-based infection system to study the effects of Streptolysin S, a secreted toxin produced by Group A Streptococcus, on keratinocytes is described. This system can be readily applied to the study of other secreted bacterial proteins on various host cell types during infection.

Many bacterial pathogens secrete potent toxins to aid in the destruction of host tissue, to initiate signaling changes in host cells or to manipulate ...

Visualizing the Effects of Sputum on Biofilm Development Using a Chambered Coverglass Model

Biofilms consist of groups of bacteria encased in a self-secreted matrix. They play an important role in industrial contamination as well as in the development and persistence of many health related infections. One of the most well described and studied biofilms in human disease occurs in chronic pulmonary infection of cystic fibrosis patients. When studying biofilms in the context of the host, many factors can impact biofilm formation and development. In order to identify how host factors may affect biofilm formation and development, we used a static chambered coverglass method to grow biofilms in the presence of host-derived factors in the form of sputum supernatants. Bacteria are seeded into chambers and exposed to sputum filtrates. Following 48 hr of growth, biofilms are stained with a commercial biofilm viability kit prior to confocal microscopy and analysis. Following image acquisition, biofilm properties can be assessed using different software platforms. This method allows us to visualize key properties of biofilm growth in presence of different substances including antibiotics.

This protocol describes the visualization of biofilm development following exposure to host-factors using a slide chamber model. This model allows for direct visualization of biofilm development as well as analysis of biofilm parameters using computer software programs.

Biofilms consist of groups of bacteria encased in a self-secreted matrix. They play an important role in industrial contamination as well as in the ...