Progress 09/01/15 to 08/31/18
Outputs
Target Audience:The target audiences are oyster hatcheries, currently located in the Pacific Northwest (Oregon, Washington states). However, we will eventually expand to Atlantic and potentially international oyster hatcheries. Our current efforts have been to develop a product that PNW oyster hatcheries with Vibrio spp. contamination can use in their hatcheries to reduce Vibrio infections and increase oyster larval survivorship. Our website http://intralytix.com/index.php?page=news&id=75 contains information on this project, where hatcheries and others can view informational videos on how phages work, and our current projects. In addition, Dr. Gary Richards at ARSUSDA has been in communication with oyster hatcheries about their issues and they have expressed interest in the Phage preparation. One oyster hatchery atRutgers Universityin Cape May, NJ is interested in performing a trial with the phage preparation in 2019 and discussions are currently ongoing. Changes/Problems:It was originally conceived that Aim 1would be straightforward and would provide results quickly and easily. The idea was to use methods already available from the literature to extract the phage DNAs and to sequence them. Unfortunately, previously published methods were not generally effective with these phages, possibly due to different structural characteristics of phages obtained from ocean depths of up to 900 meters. Since conventional methods to accomplish this aim were ineffectual, and a commercial kit to extract phage DNA was not effective, a multifaceted approach was required through the development of novel new techniques to extract and sequence the phages.Those approaches involved the: 1) development of a modified viral DNA extraction method, 2) evaluation of DNA library preparation procedures, 3) sequencing of the phage genomes, 4) sequencing of host vibrios used to propagate the phages, 5) assembly of the genomes, and 6) bioinformatics analysis of the genomes. Development of a modified DNA extraction method. Initially, a literature search was conducted to identify methods successfully used to isolate phage DNA. The effort identified several potential methods which were subsequently evaluated. The first method utilized a commercial phage DNA extraction kit using protocols specified by the manufacturer. Results were disappointing, with very poor DNA quality and quantity. Another method utilized a polyethylene glycol (PEG) phage precipitation step using PEG 8000, however phage precipitation was poor. Ultracentrifugation at 100,000 x g for periods up to 18 h at 4°C failed to pellet the phages as most of the phages remained in the supernatants. It was determined that the seawater in which the phages had been enriched interfered with the centrifugation, causing isopycnic centrifugation where the phages sought their buoyant densities within the salt solution. Consequently, salts were removed from the phage enrichments by dialysis in water for 8 h. Ultracentrifugation after dialysis effectively concentrated the phages to levels as high as 1010 plaque forming units (pfu) per ml.Consequently, phages were concentrated using dialysis for 8 h followed by ultracentrifugation for 4 h. Following phage concentration, a DNase inactivation step was applied to digest host Vibrio DNA contamination. The protocol was optimized by evaluating several DNase buffers. After the pellets were dried, they were resuspended in nuclease-free water and the DNA was allowed to dissolve for several hours at 4°C. DNA concentration and purity were determined on a NanoDrop Light (Thermo-Fisher, Waltham, MA) spectrophotometer and later on a Quibit 2.0 fluorometer (Thermo Fisher). Initial results showed poor DNA recoveries and DNA quality. Further investigation demonstrated that the DNase heat inactivation step damaged the phage capsids allowing the premature release of phage DNA before the DNase had been fully inactivated. This obstacle was unexpected and demonstrated some differences in the phages used in this study compared to previous phage studies. Clearly the integrity of these phages was compromised by elevated temperatures, which we attribute to likely structural differences of these deep-water phages. It should be noted that most of the phages were obtained from the hatchery when the source water was from 900 m deep and temperatures were <10°C. Only a few phages were isolated when the hatchery source water was changed to a shallower source (674-meters deep). These differences necessitated the development of an alternative DNase treatment procedure. After thorough investigation, a heat-sensitive DNase was obtained and successfully used in this step of the extraction to prevent premature phage lysis. The resulting DNA after purification was of much higher purity and concentration. Bioinformatic analysis of the genomes. NCBI BLAST searches were performed by David Needleman at the Nucleic Acids Core Facility at USDA, ARS in Wyndmoor, PA, at both the nucleotide level and at the amino acid level to identify one or more phage sequences from the many contigs in the FASTA files. A BLAST search of phage 6B revealed a possible prophage integrase (E = 8.59e-73), a Cl repressor (E =7.36e-30), and a regulatory Cll-like protein (E = 3.38e-6). The low E value for the Cll-like protein was concerning and out of an abundance of caution, phage 6B was removedfrom consideration for inclusion in the final cocktail formulation. The genome sequences of phages 7B and 11A did not show any homology to prophage regulatory components and therefore were considered for use in VTP-200. The genome of the more recently isolated phage 28A, which infects a broad array of V.coralliilyticus strains, was also clear of any deleterious genes and was considered for inclusion in the final VTP-200 phage cocktail (see specific aim 2). Purifying the Phage Cocktail.Preliminary experiments conducted in 1 ml wells found that the phages greatly reduced larval mortality from V.coralliilyticus infection, but the larvae would stop swimming and feeding immediately after the phage cocktail was added. The larvae remained motionless for up to 48h before regaining motility and resuming feeding. This resulted in a shorter shell length compared to the controls 48h after exposure to the phages (Figure 1). Any phage concentration high enough to reduce larval mortality would cause the larvae to stop swimming. The effect lasted for a shorter duration and had a lesser effect on shell length in 6-day old larvae than 2-day old larvae. It was hypothesized that cellular debris from the production of the phages such as metalloprotease or lipopolysaccharide were contaminating the final phage cocktail. OSU tried several methods of removing these putative cellular contaminants from the phage cocktail by filtering it through charcoal or a variety of clays but found that these methods greatly reduced the titer of the phage cocktail and were not effective. However, the effect could be eliminated, without affecting the phage titer, by performing dialysis with 100 kDa pore-size membranes. Shortly after discovering that dialysis could eliminate the toxicity of the phage cocktail, Intralytix sent OSU phages that had been purified with tangential flow filtration (TFF) (see Specific Aim 3). The TFF purified phage cocktail had no visible negative effects on larval motility or performance even at a concentration of 2 x 108 plaque forming units (PFU)/ml. The purified phages were more effective at reducing mortality than the crude phages. What opportunities for training and professional development has the project provided?While this project was not intended to train, much of the work at Oregon State University has been carried out by a graduate student who is being trained through this project. How have the results been disseminated to communities of interest?Dr. Gary Richards from USDA has presented to the National Shellfisheries Assocation, and was part of a Seafood Safety Workshop. Both of these events disseminated information to members of the community with interest in oyster health. Presentations: Presentation: Richards GP. 2017. Reduction of Vibrio coralliilyticus and Vibrio tubiashii-induced mortalities of larval oysters using bacteriophage therapy. National Shellfisheries Association, 109th Annual Meeting, Knoxville, TN, March 26-30, 2017 Richards GP. 2017. Control of enteric virus and Vibrio contamination of shellfish using novel approaches. Seafood Safety Workshop: Consumer Awareness on Seafood Safety and Public Health. Delaware State University, January 6, 2017. Additionally, Dr. Gary Richards has published a paper as part of the efforts of the project: Richards, GP, Kingham B, Shevchenko O., Watson MA, Needleman DS. Complete genome sequence of Vibrio coralliilyticus RE22, a marine bacterium pathogenic toward larval shellfish. Microbiology Resource Announcements. 7:e01332-18 https://doi.org/10.1128/MRA.01332-18 There was an additional presentation at the Hatfield Marine Science Center disseminating the efficacy results of these studies. Presentation citation: Madison D, Langdon C, Hase C, Richards GP, Watson MA, Soffer N, Li M, Sulakvelidze A. 2018. Bacteriophage significantly reduce larval oyster mortality caused by Vibrio coralliilyticus. Hatfield Marine Science Center Markham Symposium, Newport, OR, June 21, 2018. What do you plan to do during the next reporting period to accomplish the goals?
Nothing Reported
Impacts
What was accomplished under these goals?
Specific Aim 1.Phase II studies were designed to further characterize these phages in order to formulate an efficacious treatment to reduce larval mortalities in the hatcheries.To accomplish this, phage sequencing was required to show that the phages were lytic and not lysogenic.Since phages to be used in subsequent therapeutic treatments to protect larval oysters in hatcheries must receive approval for use from various State and/or Federal agencies, approvals are often dependent on demonstrating the absence of lysogenic traits.For that reason, sequencing and subsequent bioinformatics studies were performed to identify phages for potential commercial application.The first step toward this objective was to extract phage DNA.Unfortunately, previously published methods were not generally effective with these phages.Since conventional methods to accomplish this aim were ineffectual, and a commercial kit to extract phage DNA was not effective, a multifaceted approach was required through the development of novel new techniques to extract and sequence the phages.Those approaches involved the: 1) development of a modified viral DNA extraction method, 2) evaluation of DNA library preparation procedures, 3) sequencing of the phage genomes, 4) sequencing of host vibrios used to propagate the phages, 5) assembly of the genomes, and 6) bioinformatics analysis of the genomes.Phage genomes were assembled by Gian Marco Baranzoni at the USDA, ARS, Eastern Regional Research Center in Wyndmoor, PA.All identified phage genomes were subjected to a search for possible lysogenic (prophage) genes using PHASTER Specific Aim 2. An initial cocktail used in some of the studies at Oregon State University during the Phase II project utilized three phages (6B, 7B and 11A).Since sequencing and bioinformatics later identified phages 6B as potentially lysogenic, it was removed from the cocktail. In a search for phages to broaden the host specificity of the cocktail, additional seawater samples were obtained directly from the Natural Energy Laboratory of Hawaii Authority. They originated from 900-, 674-, and 20-meter-deep pipelines.One phage (28A) with broad host specificity was obtained from the shallow pipeline and was sequenced. While that work was underway, wecompleted and published a paper showing that three strains of V.coralliilyticus that are known coral pathogens also cause high mortalities in larval Pacific oysters. Those strains are OCN008, OCN014 and the ATCC type strain of V.coralliilyticus BAA-450. All phages were tested to determine if they infected these three newly recognized larval oyster pathogens. Only the latest Hawaiian strain (phage 28A) was capable of infecting and killing them.It was also lytic toward additional V.coralliilyticus strains not infected by any of our other phage isolates.By adding this phage to the cocktail, the new three-phage candidate cocktail contains phages against all eight V.coralliilyticus strains in the USDA culture collection and one of two V.tubiashii strains. The strains of V.coralliilyticus infected by the phages consist of ATCC BAA-450, ATCC 19105, 09-105-8, RE22, RE90, RE98, OCN008 and OCN014. In addition, two phages in the cocktail are infectious toward V.tubiashii ATCC 19106. Unfortunately, phage 28A was only added to the cocktail recently (after characterization, host specificity testing, sequencing and bioinformatics showed it to be highly desirable), so there is no data available on the efficacy of this particular cocktail to eradicate all the V.coralliilyticus strains listed above in a hatchery setting. Specific Aim 3. The component monophages are prepared using an aerobic fermentation process in animal-product free media. For each monophage, the host strain is grown to a target OD600, at which point the culture is infected with the monophage at a previously determined MOI (multiplicity of infection; the ratio of phage to bacteria) and the combination is incubated with aeration and mixing. The suspension is clarified by removal of bacteria by tangential-flow filtration. Following the initial filtration, the monophage is concentrated, washed with buffer, then sterilized using filtration. The individual monophage lots are analyzed to ensure each meets the release specifications before it can be used to prepare the final phage cocktail. Specific Aim 4.The initial research focused on developing a larval infection assay that would result in consistent levels of larval mortality. This work built off of previous work conducted by ARS in one ml wells of 24-well plates. OSU tested a range of V.coralliilyticus concentrations and found that adding 6.4 x 104 colony forming units (CFU)/ml of seawater consistently resulted in >99% mortality of larvae within 48h. These experiments were initially carried out in one ml wells but were scaled up to 1L, 10L, and 100L tanks. The results were consistent for all volumes tested. The effects of early exposure to phage and V.coralliilyticus were assessed by scaling up to 10 L buckets and conducting a trial that lasted 24 days. Two-day post fertilization larvae were simultaneously exposed to V.coralliilyticus and a cocktail containing phages 6B, 7B and 11A. Mortality from the initial infection was assessed 48h after inoculation. When V.coralliilyticus was added to larvae without phages, >99% of the larvae died within 48h, but when V.coralliilyticus was added to larvae with phages, the phages totally eliminated larval mortalities.The larvae that had been exposed to phage, even in conjunction with V.coralliilyticus, had a significantly higher settlement rate than the larvae-only controls. Research on phage application techniques were investigated.The method of applying the phage cocktail directly to the larval culture water (as applied in the assays reported here) would, for a commercial-sized hatchery, require a very high titer of the phage cocktail due to the volume of the tanks. The cost to produce enough phages to effectively treat a commercial hatchery in this manner could be prohibitive to the successful commercialization of the phage cocktail. Experiments carried out using phages 6B and 11A in the spring of 2018 focused on reducing the number of phages required to protect larvae by adding the phages to larvae that were concentrated in a small volume (1-2 liters) of seawater rather than adding the phages directly to the large-volume culture tanks. Four different variations of this experiment were carried out; however, none of them were successful. Concentrating the larvae appeared to increase stress, leaving them more susceptible to V.coralliilyticus infection even in the presence of phages. Specific Aim 5.Intralytix has determined that rules and regulations regarding discharge of aquaculture water is regulated locally (on a state level).Due to the nature of oyster larvae hatcheries, these facilities do not meet thedefinition of a concentrated aquatic animal production facility and therefore are not pursuant to 40 CFR Part 122.24 permit requirements.However, each aquaculture facility needs to follow local EPA rules, and apply for permits associated with their local laws.These also depend on whether the facility treats its water effluent before discharging (or if it discharges to the local sewage system).
Publications
- Type:
Journal Articles
Status:
Accepted
Year Published:
2018
Citation:
Richards, GP, Kingham B, Shevchenko O., Watson MA, Needleman DS. Complete genome sequence of Vibrio coralliilyticus RE22, a marine bacterium pathogenic toward larval shellfish. Microbiology Resource Announcements. 7:e01332-18 https://doi.org/10.1128/MRA.01332-18
- Type:
Conference Papers and Presentations
Status:
Other
Year Published:
2018
Citation:
Madison D, Langdon C, Hase C, Richards GP, Watson MA, Soffer N, Li M, Sulakvelidze A. 2018. Bacteriophage significantly reduce larval oyster mortality caused by Vibrio coralliilyticus. Hatfield Marine Science Center Markham Symposium, Newport, OR, June 21, 2018.
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Progress 09/01/16 to 08/31/17
Outputs
Target Audience:The target audiences are oyster hatcheries, currently located in the Pacific Northwest (Oregon, Washington states). However, we will eventually expand to Atlantic and potentially international oyster hatcheries. Our current efforts have been to develop a product that PNW oyster hatcheries with Vibrio spp. contamination can use in their hatcheries to reduce Vibrio infections and increase oyster larval survivorship. Our website http://intralytix.com/index.php?page=news&id=75 contains information on this project, where hatcheries and others can view informational videos on how phages work, and our current projects. In addition, Dr. Gary Richards at ARS-USDA has been in communication with oyster hatcheries about their issues and they have expressed interestin the Phage preperation. Changes/Problems:Due to the seasonal timing of oyster larvae availability, we were only able to conduct Vibrio infections starting in late June. Therefore, to finish conducting all the planned experiments, we have asked for and received a 12 month extension to increase the scale of the infection studies (from 1 ml to 100 liters+), and to be able to more fully quantify the results of these experiments. Infection experiments at Oregon State University are expected to run into November 2017, with many more months to analyze the data, finalize the product and prepare for commercialization. However, despite the delay in experiments, the results have thus far been very encouraging, and we expect the scale up experiments to at least 100-liter volumes to show similar results. Another delay in our project was due to the sequencing efforts. The original sequences were not as high quality as we desired for full analysis. Therefore, sequencing was repeated for all the phages and we are currently re-analyzing those sequences. What opportunities for training and professional development has the project provided?While this project was not intended to train, much of the work at Oregon State University has been carried out by a graduate student who is being trained through this project. How have the results been disseminated to communities of interest?Dr. Gary Richards from USDA has presented to the National Shellfisheries Assocation, and was part of a Seafood Safety Workshop. Both of these events disseminated information to memebers of the community with interest in oyster health. Presentations: Presentation: Richards GP. 2017. Reduction of Vibrio coralliilyticus and Vibrio tubiashii-induced mortalities of larval oysters using bacteriophage therapy. National Shellfisheries Association, 109th Annual Meeting, Knoxville, TN, March 26-30, 2017 Richards GP. 2017. Control of enteric virus and Vibrio contamination of shellfish using novel approaches. Seafood Safety Workshop: Consumer Awareness on Seafood Safety and Public Health. Delaware State University, January 6, 2017. What do you plan to do during the next reporting period to accomplish the goals?For Specific Aim 1, Intralytix is currently further analyzing the new set of genomicsequences to confirm all findings, and then Aim 1 will be complete. For Specific Aim 4. Oregon State University is currently analyzing data from their 24 day, 10 liter bucket experiments. Also, at this point, the phage cocktail has only been tested against Vibrio strain RE98 in inoculation experiments. The next experiment taking place at OSU (in the coming weeks) will use both Vibrio hosts (RE98 and RE22), and on a larger scale (100 Liter volume) that more accurately reflects hatchery conditions. For Specific Aim 5. Intralytix has determined that rules and regulations regarding discharge of aquaculture water, is locally regulated (on the state level). Due to the nature of larval oyster hatcheries, these facilities do not come under 40 CFR Part 122.24. However, each aquaculture facility needs to follow local EPA rules, and apply for permits associated with their local laws. Intralytix will aid any aquaculture facility using phage treatment VTP-200 with the paperwork necessary, depending on whether they are in Oregon, Washington or any other state. These also depend on whether the facility treats its water effluent before discharging (or if it discharges to the local sewage system). Lastly, we are exploring a method to apply phages to the oyster larvae directly, rather than applying them to the water, which involves concentrating the oyster larvae onto mesh containers (we have observed this done at Whiskey Creek Hatchery: they do this on a daily basis) and dipping into a bucket of concentrated phage before adding them back to their water tanks (10,000+ gallon tanks). This will both reduce the volume of phages needed per hatchery, and avoid any release of phages into the environment.
Impacts
What was accomplished under these goals?
Specific Aim 1 Characterize the genomic makeup of V. tubiashii- and V. coralliilyticus-specific phages for potential inclusion in a commercial phage preparation Specific Aim 1 was carried out by Dr. Gary Richards from USDA-ARS. Considerable effort has been expended to extract and sequence the DNA of six phages (four of those used in the final cocktail). Genomic sequencing of six phages, including the four used in the final VTP-200 cocktail has been successfully performed at the USDA, ARS laboratory in Wyndmoor, PA, using the Illumina MiSeq sequencing platform. They are 184,656 base pairs (bp), 205,414 bp, 39,562 bp, 30,208, 192,704 bp and 136,241 bp in length, respectively. Of these phages sequenced, phages, 28A, 7B and 6B will likely be used in the final cocktail, with the possibility of 11a as well. These likely represent the entire phage sequences, since myoviruses and siphoviruses (the types of phages isolated) usually contain between 30,000 and 300,000 bp. Subsequent NCBI BLAST searches were performed by the Nucleic Acids Core Facility at USDA, ARS in Wyndmoor, PA, at both the nucleotide level and at the amino acid level to identify one or more phage sequences from the FASTA files. Multiple phage proteins were detected in the sequences. Phages were also re-sequenced for higher quality analysis. Of these new sequences,Intralytix ran the sequences through Virulence Finder-1.5 (https://cge.cbs.dtu.dk/services/VirulenceFinder/) to confirm that no undesirable virulence genes were present in the final cocktail (ie antibiotic resistance, toxins etc). Specific Aim 2 Formulate a second generation, candidate commercial phage cocktail (VTP-200) New phages were isolated for VTP-200 to increase the target range of the current cocktail to include V. coralliilyticus RE22. First, these phages were isolated from seawater from the Natural Energy Laboratory of Hawaii Authority in Kailua-Kona, Hawaii, from deep ocean pipelines (900-meters and 600-meters deep) and from surface water. This location provided the source water from which all of our phages against Vibrio coralliilyticus and Vibrio tubiashii were previously isolated and used to formulate the preliminary cocktail VTP-100. No other sources of seawater from coastal areas along the continental U.S. have contained phages against V. coralliilyticus. In the current evaluation of seawater, a phage against RE22 was successfully isolated from the sample taken from the 20-meter deep NELHA pipeline after enrichment. In addition, this new phage also shows broader host specificity than previous phages, since it is capable of infecting and killing a previously uninfectable strain of V. coralliilyticus known as RE90. This phage has been characterized, and confirmed as a lytic phage that shows high titers during production and ability to kill the targeted Vibrio strains, and will be part of the final VTP-200 phage cocktail. Depending on how the addition of this new phage affects the current RE98-targeting cocktail, another phage may be removed or added. Specific Aim 3 Scale-up the manufacture of the candidate phage cocktail Intralytix has had good success scaling up the manufacture of each of the individual candidatephages. Originally, the phages were grown on filtered natural seawater or full strength marine broth. The maximum titers achieved were approximately 1x109 PFU/ml per monophage, requiring essentially all of the produced phage to be included in a full strength cocktail (final concentration 1x1010 PFU/ml). Intralytix has improved its cocktail formulation by testing multiple scale up conditions, including ratio of media to seawater, MOI (multiplicity of infection), temperature, shaking speed (aeration), time of incubation before infection and optical density of the host before infection. By determining the optimal combination of production conditions, we have scaled up production of each monophage in the cocktail and increased the manufacturing titers up to 100 fold (from 1x109 pfu/ml to 1x1011 pfu/ml). At minimum, each monophages can now be grown to 1x1010 pfu/ml. In addition, we have been able to grow each monophage in artificial seawater with minimal marine broth, and now include a tangential flow filtration step to clarify the monophages, exchange the buffer from its growth media to pure artificial seawater and reduce the endotoxin concentrations from ~500,000 EU/ml to <50,000 EU/ml at the 10X concentration; for reference, our consumable bacteriophage products are acceptable at <250,000 EU/ml at the 10X concentration. The reduction of endotoxins has resulted in higher survivability, faster growth, and enhanced motility of the oyster larvae during in vivo experiments. Specific Aim 4 Perform efficacy studies to determine the optimal treatment regimen for reducing V. tubiashii- and V. coralliilyticus-associated mortality of larval oysters by at least 50% Since our last report, OSU has conducted additional, and multiple trials with candidate VPT-200 and controlled Vibrio infections (RE98 strain specifically).In addition to testing the phage cocktail made with the new QC protocols, the "old" phage that was made with the original protocols (i.e., in filtered natural seawater and without endotoxin removal) was also compared to the new phage cocktail. In this trial, multiple phage concentrations were tested and it was determined that 106 PFU/ml reduced mortality from V. coralliilyticus strain RE98 by 60%, while 107 and 108 PFU/ml reduced mortality by >90% . The 107 and 108 PFU/ml phage + vibrio treatments were not significantly different from the larvae only controls (Tukey HSD). This shows that 107 PFU/ml is sufficient to greatly reduce mortality from 104 Vibrio cells/ml. After this small-scale experiment, two parallel experiments using phages at 107 PFU/ml and Vibrio infections at 104 cells/ml were carried out in 10 liter buckets for 24 days. The first experiment included a single infection and single phage application, the other experiment include multiple infection events and phage applications. The results from these long term trials are still being analyzed, but thus far it was determined that for the single infection and phage application experiments mortality was highly decreased with the addition of phages to the water, from 100% mortality with Vibrio added but no phage treatment to only ~15% mortality with the phage treatment after 4 days (which is not significantly different than control oyster larvae with no Vibrio added). In addition, zero oyster larvae infected with Vibrio survived long enough to mature and settle after 24 days, but with the addition of phages (and Vibrios)there was no significant reduction of oyster settlement compared to controls without any vibrio infections, and/or just phage additions with no bacterial infections. The current phage formulation has successfully reduced mortality by >90% during the critical 48 hour growth stage, with no long term effects in settlement/ maturity of oyster larvae after 24 days.
Publications
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Progress 09/01/15 to 08/31/16
Outputs
Target Audience:The target audiences are commercial oyster hatchery operators. Intralytixhas visited a potential consumer, one of the largest oyster larvae hatcheries in the Pacific NorthWest, USA. At the oyster hatchery,Intralytix and partners discussed the potential use of the bacteriophage product in a commercial setting, such as likely methods for easily applyingproduct, seasonal usage, prevention vs. treatment and other aspects of the potential product. Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided?Dr. Gary Richards (ARS, Dover, DE) and Intralytix travelled to Oregon State University to provide training demonstrations on plaque assay techniques to OSU students and staff. A representative from Intralytix attended the SBIR training workshop in Washington DC in mid December, 2015. The lectures, workshops were profesional development and training experianaces. There were also on going webinars and training via an the LARTA network and assigned mentor. How have the results been disseminated to communities of interest?Information of the grant and its efforts are pubslihed on the Intralytix website:http://intralytix.com/index.php?page=news&id=75 What do you plan to do during the next reporting period to accomplish the goals?For Aim 1,more phage sequences will sequenced andcharacterized by the USDA -ARS and Intralytix, these will be submitted to GenBank when finalized. For Aim 2,Intralytix will formulate an optimal cocktial using its proprietary Phage Selector next. For Aim 3, Intralytix will use its expertise to scale up the phage production. Preliminary conditions for growing phages have been determined. For Aim 4, Oregon State University will test the final product in a replicated commerical setting. They are currently optimizing commercial hatchery Vibrio infectionconditions. Aim 5 will be achieved once the experiments are completed and Intralytix will then have data required to subsequently assemble regulatory packages for commercialization.
Impacts
What was accomplished under these goals?
For Aim 1,genomes of 4 promising Vibrio infectingbacteria were sequenced and the genomes characterized. More specifically, sequencing of four of the six phage DNAs has been successfully performed at the USDA, ARS laboratory in Wyndmoor, PA, using the Illumina MiSeq sequencing platform. Assembled sequences (contigs) were analyzed using VirSorter on iPlant Discovery Environment (https://de.iplantcollaborative.org.de). VirSort results identified sequences from DNA extracts from four phages (designated 5A, 6B, 7B and 11A) as likely phage sequences. They are 205,414 base pairs (bp), 192,704 bp, 207,758 bp and 192,704 bp in length, respectively. Subsequent NCBI BLAST searches were performed by the Nucleic Acids Core Facility at USDA, ARS in Wyndmoor, PA, at both the nucleotide level and at the amino acid level to identify one or more phage sequences from the FASTA files. The phage sequences are being transferred to Intralytix for further bioinformatic analyses, and be prepared for submission to GenBank next. For Aim 2, new bacteriophages were selected from seawater obtained from deep ocean water (600-900 meters deep). Some of these phages were characterized (description above). For Aim 4,consultants at Oregon State University (Dr. Chris Langdon)are currently doing efficacy studies. Thus far, they have been able to replicate mortality rates equivalent to those previously experienced at a commercial hatchery with a Vibrio infection ( ie up to 100%) on a micro-level and have increased the scale to 1 liter batches. There were originally inconsistenciesachieving this mortality rate, as the addition of ampicillin reduced pathogenicity, however removing ampicillin and using 104colony forming units (CFU)/ml has yielded consistent, replicable results. Additionally, they determined that inoculating oyster larvae with Vibrios that are harvest in the exponential growth stage also increased pathogenicity, making them more suitable for efficacy studies. The next step is to add the bacteriophage cocktail (VPT-200) and scale up to commercial levels.
Publications
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