NEW TREATMENTS FOR AMERICAN FOULBROOD: USING THE MICROBE`S BIOLOGY AGAINST IT

• Sponsoring Institution: National Institute of Food and Agriculture
• Project Status: COMPLETE
• Funding Source: AFRI COMPETITIVE GRANT
• Reporting Frequency: Annual
• Accession No.: 0224189
• Grant No.: 2011-67013-30169
• Cumulative Award Amt.: $333,276.00
• Proposal No.: 2010-03755
• Project Start Date: Feb 1, 2011
• Project End Date: Jan 31, 2015
• Grant Year: 2011
• Program Code: [A1111]- Plant Health and Production and Plant Products: Insects and Nematodes

Recipient Organization
UNIVERSITY OF NEVADA
4505 S Maryland Pkwy
LAS VEGAS,NV 89154

Performing Department
School of Life Sciences

Non Technical Summary
Honey bee pollination is crucial to ensuring sustainable food supplies worldwide, yet honey bee abundance is decreasing worldwide due to a variety of pests and diseases. American foulbrood is a larval honey bee disease caused by the spore forming bacteria P. larvae. Antibiotics can cure the active infections, but the spores are easily tranferred between colonies and can remain for decades to re-infect generations of bees. American foulbrood is typically highly regulated requiring the use of confirmatory diagnostics and often resulting in forced incineration of the bees and equipment making it a costly disease. We will attack the microbe using its own biology against it and take advantage of that biology and that of the bees themselves to protect the larvae. First,we plan to identify bio-molecules that signal spore germinantion and use these compounds to cause the spores to germinate out of context, thus making them vulnerable to other treatments such as P. larvae specific lytic enzymes that break open cell walls which we will derive from viral phage that attack P. larve. Second, we plan to protect the larvae with anti-germination signaling compounds and antimicrobial peptides found in non-susceptible adult bees. As a whole, this approach combines several novel routes of control to produce treatments that can protect bees as well as remove the infectious spores from the hive without the risk of resistance posed by current antibiotic treatments or contamination of the hive and its products.

Animal Health Component

40%

Research Effort Categories

Basic

60%

Applied

40%

Developmental

(N/A)

Classification

Knowledge Area (KA)	Subject of Investigation (SOI)	Field of Science (FOS)	Percent
211	3010	1100	75%
211	3010	1020	25%

Knowledge Area
211 - Insects, Mites, and Other Arthropods Affecting Plants;

Subject Of Investigation
3010 - Honey bees;

Field Of Science
1100 - Bacteriology; 1020 - Physiology;

Keywords

foulbrood

paenibacillus larvae

honey bee

phage

antimicrobial peptides

microbial germination

Goals / Objectives
We plan to produce new bio-rational control agents for foulbrood, a larval honey bee disease caused by the microbe Paenibacillis larvae. We are going to pursue a two pronged attack on the disease first by attacking the microbe directly and second by proecting the larvae. In objective 1 we will identify bio-molecules that signal spore germinantion and use these compounds to cause the spores to germinate outside of the larval bee gut. We will also indentify compounds that inhibit P. larvae germination and feed these to larvae. As later larval stages and adult bees are not susceptible to the disease, in objective 2 we will screen the hemolymph of these stages for proteins that may provide resistance and also provide these to the younger larvae. In Objective 3 we plan to identify P.larvae specific phages (viruses that attack microbes)which will produce lytic enzymes, lysins. that can be used to attack the microbes we cause to germinate using the germination signalling molecules. These lysins have the benefit of being specific for the microbe in question so unlikely to harm other organisms-particularly beneficial gut microbes of the bees. All of the above experiments will occur simultaneously during the first two and a half years of the grant. Finally, during year 3 for objective 4 we will test these compounds on larval bees raised in vitro as a first step toward developing a combined treatment. At the end of the project we will have produced germinant solutions and lysins to attack the microbe and antigerminant solutons and proteins to protect the honey bee larvae.

Project Methods
In Objective 1 we will use a step-wise approach to identify compounds from a defined media that trigger germination,using kinetic analyses based on optical density of the bacteria containing media. Germination rates can then be used to determine the order of germinant binding, and the mechanism of germination receptor activation even without knowing the identity of the receptor. In turn, this information can be exploited to determine the most likely molecular pathways that can be used to disrupt germination. After identifying germinants and anti-germinant compounds we will then prepare and screen germinant analogs to determine functional groups necessary for P. larvae spore germination activation and inhibition.In Objective 2 we will measure the abundance of antimicrobial peptides (AMPs; already identified in bees) and pro-AMPs in the hemolymph of adults and larvae.. Equal volume samples of diluted lymph will be analyzed by HPLC-MS using an ABI LC/MS Q-trap in reverse-phase mode. Concentration of the different AMPs and their precursors will be assessed by comparison with known standards. Second we will measure the susceptibility of vegetative P. larvae to and full-length and processed AMPs in vitro. Wild type P. larvae cultures will be diluted to A600=2x105 cells/ml in MYT medium and incubated with increasing concentrations of the AMP in 96 well plates. The lowest concentration needed to prevent bacterial growth will be recorded as the minimal inhibitory concentration (MIC) for each AMP. Third, we will determine if any of these AMPs inhibit spore germination or kill actively germinating spores of P. larvae in vitro. Increasing concentrations of AMPs will be added to sporulated and germinating P. larvae and changes in optical density will be used to determine efficacy of inhibition. In Objective 3 we will identify P. larvae specific phage from soil around hives; sequence one or more of these phage to identify te lysin producing genes and produce the lysins. In objective 4 we will raise larve in vitro on the well established larval diet and test these compounds (germinants, anti-germnants, AMPs and phage lysins) on larvae at susceptible and non-susceptible stages against untreated controls. We will measure survival, expression of honey bee immune response genes, and hemolymph antimicrobial proteins to assertain the effectiveness of these potential treatments to protect larvae from infection. We will also test the ability of the germinants to cause germination of spores on hive components by spreading those components with spores, allowing them to dry and spraying them with germinant solutions. Following this we will clean with 10% bleach, 70% ethanol or quaternary salt detergents and measure spores still on the equipment relative to distilled water treated controls. We will repeat this with the AMPs, lysins and combinations of our treatments.

Progress 02/01/11 to 01/31/15

Outputs
Target Audience: We have multiple target audiences; other scientists, beekeepers and the general public. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided? Two students, Diane Yost and Jasmin Khilnaniobtained MS degrees (both women, one African American) and one student, Israel Alvarado,will finish his PhD in the summer of 2015 (he is also Hispanic). More than 14 undergraduate students have been trained in research and microbiology. Lucy LeBlanc was awarded an NSF Research Experience for Undergraduates Fellowship for spring of 2014 and an American Society for Microbiology (national competition) Undergraduate Research Fellowship in 2015to work on her lysin project and will also attendthe Capstone course which is a pre-meeting event to develop professional skills. Carolyn Chang was awarded an NSF Research Experience for Undergraduates Fellowship for spring of 2015 to isolate new phages using an alternative host to determine phage host susceptibility when isolated on a Paenibacillus larvae strain from the subspecies pulvifaciens (now ERIC groups III and IV.) Her poster will be publically presented Friday 4/17/15. How have the results been disseminated to communities of interest? All members of the team have participated in outreach to beekeepers and to the public about honey bees and their diseases in general and this work in particular. See the products section for the scientific presentations at conferences and publications to disseminate the research to the scientific community. Outreach to bee keepers in PA, NV and IA describing the problem of AFB and our approach resulted in environmental samples of wax, soil, bees, propolis, royal jelly, and one scale sample from an infected hive. Israel Alvarado from the Elekonich and Abel-Santos labs, presented a talk on crop pollination and bee diseases to 3rd graders at Tartan Elementary School in North Las Vegas. Dr. Wing described our work during an invited seminar in the College of Life Sciences at Brigham Young University in March 2011. This college is an active participant in the "Phage Hunters Program" sponsored by Howard Hughes Medical Institute, and our phage work generated considerable interest. Dr. Elekonich described our work in an invited seminar at Georgetown University and in a talk at the National Science Foundation. Additionally, Dr. Amy and undergraduate student Lucy LeBlanc havegiven presentations to numerous grade school, middle school and high school students have heard the story of AFB, honeybees and our approach to saving them. Lucy made up a 'phage dance' for the elementary school children which they quite enjoyed as they danced around the room. We have also received some coverage by the local news media: In the Las Vegas Review Journal: www.lvrj.com/news/unlv-researchers-fight-disease-affecting-honeybees-161759595.html The Las Vegas Weekly: http://www.lasvegasweekly.com/news/2012/jul/12/more-human-microbes-are-essential-part-our-worldan/ The Las Vegas Sun: http://www.lasvegassun.com/news/2012/jul/12/more-human-microbes-are-essential-part-our-worldan/ What do you plan to do during the next reporting period to accomplish the goals? Nothing Reported

Impacts
What was accomplished under these goals? Impacts: We identified germination receptors, germinants and germination inhbitors of P. larvae.We have identified 5-chloroindole as strong inhibitor of spore germination, cell growth, and AFB disease development and it has the lowest IC50 value, is inexpensive, and highly soluble in water. We tested all the honey bee anitmicrobial peptides and found that the defensins were the most effective against P. larvae. We identified phage with high host specificity to P. larvae with broad efficacyand developed a method of using phage to treat active infections which was tested in colonies andsubmitted for patent. Activities: Previously we found P. larvaespores germinated rapidly in response to L-tyrosine and uric acid. Compounds with similar chemical structure toP. larvaegerminants did not stimulate germination. Spore germination was not observed in complex media or in an artificial medium containing universal germinants. The germination profile of environmentally derived spores was identical to spores from a typed strain suggesting that the type strain remains indicative of the wild type bacteria. Indole and phenol, strongly inhibited spore germination. Ten of the 40 indole and phenol analogs tested were strong inhibitors of P. larvae B3685 spore germination. Survival of in vitro reared honey bee larval survival was significantly higher for larvae fed indole analogs (36-75%) than spores (22%). The highest survival percentages were observed in larvae fed with 5-bromoindole (75%) and 5-chloroindole (65%). We have identified 5-chloroindole as strong inhibitor of spore germination, cell growth, and AFB disease development and it has the lowest IC50 value, is inexpensive, and highly soluble in water. Larval exposures assays were conducted using 0.125, 0.25, 0.5, 0.75, or 1.0 mM 5-chloroindolein larval diet. Although there were no statistically significant differences in survival between larvae fed different doses of 5-chloroindole and those fed the control diet,with the 1.5 mM 5-chloroindole treatment we observed 100% mortality 24 hours post treatment. Mortality was reduced to approximately 5% cumulative at concentrations below1 mM 5-chloroindole. During sporulation germination receptors are incorporated within the inner membrane to allow spores to germinate, under favorable environmental conditions. The genome of P. larvae strain B-3650 contains five loci with gene sequences that are similar to known B. subtilis ger and prkC germination receptors, but have not been functionally characterized. We detected the expression of gerKA3 and gerKA4 germination receptor mRNAs during sporulation. Furthermore, no germination receptor mRNAs were detected in exponentially growing cultures. Two approaches were developed toassay the antimicrobial activity of honeybee AMPs against P. larvae the Zone of Inhibition Assays and the 96-well plate format. We characterized five individual honeybee antimicrobial peptides (AMPs), apidaecin, abaecin, hymenoptaecin and defensins 1 and 2, against vegetative cells of P. larvae using in vitro assays. We found that each AMP affects the growth of P. larvae at different time points (6, 12, 18 or 24 hours). Defensins 1 and 2 have the greatest inhibitory effect, at some times greater than a 25% decrease. We tested whether the antimicrobial killing of P. larvae by defensin 2 was enhanced by the addition of one of the other honeybee AMPs used in our studies (defensin 1, apidaecin, abaecin, hymenoptaecin). We chose defensin 2 for this work, because it circulates in honeybee hemolymph and is not as well characterized as antimicrobial of P. larvae as defensin 1. A fixed number of vegetative P. larvae cells (as judged by spectrophotometry) were incubated with each of the AMPs at a final concentration of 50ug/ml for 24h. Based on our statistical analysis, none of the AMP combinations displayed more antimicrobial activity than when defensin 2 was used alone. Defensin 2 and apidaecin displayed the most antimicrobial activity Unfortunately, when the defensins were combined they displayed less antimicrobial activity than when each was used alone. We conclude that the antimicrobial effect of the defensins against P. larvae cannot be enhanced by using them in combination. To find and describe environmental bacteriophages, 157 samples were obtained fromphage, cosmetics, soil under hives, beehive materials and other environmental sources. Thirty samples were positive for at least one bacteriophage capable of growing on P. larvae strain 2605; two sources produced two phage isolates each for a total of 32. Host range data further indicated that each phage provided a unique pattern of infectivity on 11 P. larvae bacterial strains, with the exception of three pairs of identical patterns even when the phages were isolated from highly different sources. Seventeen of the bacteriophages have been selected for DNA extraction, amplification, and restriction enzyme digest profiling. Sixteen phages were imaged by TEM with 13 belonging to the Siphoviridae. Thirteen of the phages are capable of lysing at least 80% of the 10 P. larvae strains we have tested them on. Two of the phages were selected because they have the highest number of P. larvae strains that they completely lyse.One of these is also one of the phages that lyses at least 80% of the P. larvae bacterial strains. These two phages are being considered for lysin production. Isolated bacteriophages were combined to produce two different phage cocktails containing more or fewer phage types. These were administered to lab-reared honey bee larvae infected with cells and spores of P. larvae.The survival of larvae treated with the phage cocktail prior to infection increased by 70%, and was comparable with the survival rates of the phage cocktail controls. We alsotested host range of our bacteriophage on other species of Paenibacillus and on othergenera of bacteriatoevaluate the host specificity of the phage. To date, the twelve bacterial species from four other genera are not remotely susceptible to P. larvae bacteriophage. Out of the two other Paenibacillus species, only one was slightly susceptible to six of the 31 bacteriophage.These results indicate a high host specificity in the phage and that using them in a hive would most likely not negatively affect non-AFB causing bacteria. Lucy LeBlanc found sequences in the NCBI database for lysogenic phages in the one full Paenibacillus larvae sequence from which she designed primers to amplify the lysin gene and tested them on phage Xenia.She expressed the lysin gene in E coli using confirmed lysis of the P. larvae bacteria with a lysin generated in E. coli. She purified the lysin and tested it in larvae using appropriate controls; as a result of lysin therapy the larvae survived to nearly the control level.Although spore treated larvae began to die by day three of the trial, none of the lysin treated larvae died until day six. One field experiment was conducted near Bellingham, WA in summer, 2013. Improvement was seen in the hive that was highly infected with AFB, however the hive was not able to completely recover prior to the onset of winter. Removal of frames containing the worst infection and insertion of empty, new frames resulted in no visible return of disease over a two month period.

Publications

• Type: Theses/Dissertations Status: Published Year Published: 2014 Citation: Yost, Diane Gerda. Isolation and Characterization of Paenibacillus larvae Bacteriophages for Use as a Treatment of American Foulbrood Disease in Honeybees. Degree: MSin Biological Sciences, Biological Science, 2014, University of Nevada  Las Vegas URL: http://digitalscholarship.unlv.edu/thesesdissertations/2160
• Type: Theses/Dissertations Status: Other Year Published: 2015 Citation: Thesis Khilnani, Jasmin C; MS Thesis: The Effects of Honeybee (Apis mellifera) Antimicrobial Peptides on Paenibacillus larvae. In progress.
• Type: Theses/Dissertations Status: Under Review Year Published: 2015 Citation: Alvarado, Israel; PAENIBACILLUS LARVAE SPORE GERMINATION AND AMERICAN FOULBROOD DISEASE DEVELOPMENT IN HONEY BEE LARVAE PHD Dissertation
• Type: Journal Articles Status: Accepted Year Published: 2015 Citation: Ghorbani-Nezami S, LeBlanc L, Yost DG and Amy PS. 2015 Phage Therapy is Effective in Protecting Honeybee Larvae from American Foulbrood Disease. Journal of Insect Science. (Journal of the Entomological Society of America) Accepted.
• Type: Journal Articles Status: Under Review Year Published: 2015 Citation: Yost DG, Tsourkas P and Amy PS. 2015. Isolation and Characterization of Bacteriophages used in Experimental Treatments of Honeybess (Apis mellifera) infected with Paenibacillus larvae, the causative agent of American Foulbrood Disease. Journal for Microbial Biotechnology (a Journal of the Society for Industrial Microbiology).
• Type: Journal Articles Status: Other Year Published: 2015 Citation: Alvarado I, Khilnani J, Yost, D.G., Elekonich M, Abel-Santos E and Wing HJ. 2015. Comparison and adaptation of methods to aid development of treatment strategies against the honey bee pathogen Paenibacillus larvae, the causal agent of American Foulbrood disease. Journal of Microbiological Methods. Under Revision following submission.
• Type: Conference Papers and Presentations Status: Other Year Published: 2013 Citation: Yost, D. and P.S. Amy. Characterization of Bacteriophages which Infect Paenibacillus larvae, an Important Honeybee Pathogen. Abstract submitted and accepted for the American Society for Microbiology annual meeting in Denver, CO. Presentation May 2013.
• Type: Conference Papers and Presentations Status: Other Year Published: 2013 Citation: Smith, J. and Wing, H. Testing Honeybee Antimicrobial Peptides to Counter the Growth of the Causative Agent of American Foulbrood Disease, Paenibacillus larvae. AZ/Southern Nevada branch of ASM. University of Arizona, Tuscon, AZ (April 13th 2013)
• Type: Conference Papers and Presentations Status: Other Year Published: 2013 Citation: LeBlanc, L. Amy, P.S. and Yost, D.G. The Search for Bacteriophage Lysins Active against the Honey Bee Pathogen, Paenibacillus larvae. UNLV NSF REU poster presentation. August, 2013.
• Type: Conference Papers and Presentations Status: Other Year Published: 2013 Citation: Yost, D. and P.S. Amy. Characterization of Bacteriophages That Infect Paenibacillus larvae, an Important Honeybee Pathogen. ASM GM 2013 Denver, CO
• Type: Conference Papers and Presentations Status: Other Year Published: 2014 Citation: L. LeBlanc, P. S. Amy, P. Tsourkas, S. Nezami. Isolation and Characterization of a Novel Phage Lysin Active against Paenibacillus larvae, a Honeybee Pathogen. AZ/Southern Nevada branch of ASM. University of Arizona, Tuscon, AZ Won best undergraduate student poster
• Type: Conference Papers and Presentations Status: Accepted Year Published: 2015 Citation: L. LeBlanc, P. S. Amy, P. Tsourkas, S. Nezami. Isolation and Characterization of a Novel Phage Lysin Active against Paenibacillus larvae, a Honeybee Pathogen. Poster accepted for the May 29-June 2, 2015 National meeting of the American Society for Microbiology in New Orleans, LA.
• Type: Conference Papers and Presentations Status: Other Year Published: 2014 Citation: Jasmin C Khilnani and Helen J Wing. Growth Inhibition of Paenibacillus larvae Using Honeybee Antimicrobial Peptides UNLV GPSA Spring 2014 Research Forum, 29 March 2014
• Type: Conference Papers and Presentations Status: Other Year Published: 2014 Citation: Jasmin C Khilnani and Helen J Wing Growth Inhibition of Paenibacillus larvae Using Honeybee Antimicrobial Peptides Presented at 53rd Annual ASM regional branch for AZ and NV meeting, 5 April 2014
• Type: Journal Articles Status: Submitted Year Published: 2015 Citation: LeBlanc L, Tsourkas P, Amy PS. 2015. Isolation and Characterization of a Phage Lysin Active Against Paenbacillus larvae, a Honeybee Pathogen. Antimicrobial Agents and Chemotherapy (a journal of the American Society for Microbiology).
• Type: Journal Articles Status: Other Year Published: 2015 Citation: Tsourkas P, Yost DG, Krohn, A, LeBlanc L, Amy, PS. 2015. Complete genomes of eleven phages that infect Paenibacillus larvae, the causative agent of American Foulbrood disease in honeybees. Genome Announcements. Four new phage sequences will be added.

Progress 02/01/13 to 01/31/14

Outputs
Target Audience: We have multiple target audiences; other scientists, beekeepers and the general public. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided? Two MS and one PhD student continued their work. All will likely finish their degrees during the next reporting period. A total of 6 undergraduates were involved in various aspects of the project and learned both field and laboratory techniques. How have the results been disseminated to communities of interest? All members of the teamcontinue to participatein outreach to beekeepers, school childrenand to the public about honey bees and their diseases. Dr. Elekonich presented this work and other work from her lab in a seminar at the Department of Entomology at the University of Maryland, College Park. See the products list for conference presentations. All 3 graduate students presented their work at the UNLV School of Life Sciences student colloquium. What do you plan to do during the next reporting period to accomplish the goals? Objective 1: We will continue to test the ability of halide and nitro groups to enhanced indole’s activity to prevent spore germination. Furthermore, we are identifying P. larvae spore germination receptors using genetic tools. Once P. larvae spore receptors are known we can utilize a structure based strategy for the synthesis of stronger germination inhibitors. Objective 2: We plan to test honeybee hemolymphfrom adult, nurse bees and larvae at different stages, second and late third instars for presence of AMPs and inhbitory activity. We would test the hemolymph using our semisolid and liquid media assays described in last year’s report. If differences in the inhibitory activity are observed against P. larvae using hemolymph, we will send the samples for protein profile analysis to see if there are differences. Finally, we have begun preliminary testing to develop assays to test the effect of AMPs on the resulting outgrowth from germinating P. larvae spores. Objective 3: We plan to test the phage cocktail with larvae in vitro and on equipment. Additonal tests in infected hives will be carried out with cooperating beekeepers depending on availability. Objective 4: In vitro Tests combining germinants with phage to see if we can successfully use the germinant cues to cause the spores to become vegetative cells and follow up with phage to kill all those cells. During the next reporting period we will also be focusing on publishing any as yet unpublished work from all 3 prior reporting periods.

Impacts
What was accomplished under these goals? Objective 1: We previously identified triggers (L-tyrosine plus uric acid) and inhibitors (indole or phenol) of P. larvae spore germination in vitro. Because L-tyrosine and uric acid in honey bee larvae are likely in saturating concentrations, we sought to identify stronger germination inhibitors. We screened 40 indole and phenol analogs for their ability to act as antagonists of P. larvae B3685 spore germination. We hypothesized that the addition of functional groups to indole and phenol molecules would enhance their interaction with putative germination receptors. Ten of the 40 indole and phenol analogs tested were strong inhibitors of P. larvae B3685 spore germination. We found that indole analogs with electron withdrawing groups (EWG) were prevented spore germination in vitro. The half maximal inhibitor concentration (IC50) for analogs ranged between 0.02-0.55 mM. The indole and phenol analogs identified will be used to determine if inhibiting P. larvae spore germination prevents AFB disease in honey bee larvae. Objective 2: Since our last report, we have finished characterizing five individual honeybee antimicrobial peptides (AMPs), apidaecin, abaecin, hymenoptaecin and defensins 1 and 2, against vegetative cells of P. larvae using in vitro assays. We found that each AMP affects the growth of P. larvae at different time points (6, 12, 18 or 24 hours). However, overall results demonstrate that defensins 1 and 2 have the greatest inhibitory effect, at some times greater than a 25% decrease. Currently, we are testing the AMPs in different combination pairs to observe if there are any additive or synergistic effects on P. larvae growth inhibition. Objective 3: To find and describe environmental bacteriophages, 157 samples were obtained from many diverse sources including: P. larvae strains harboring prophages; various soils; wax, honey, royal jelly and propolis; cosmetics containing products derived from beehives; plants and flowers; compost; water and air samples. Of the 157 samples, 30 were positive for at least one bacteriophage capable of growing on P. larvae strain 2605 for a total of 32; two sources produced two phage isolates each. After isolation, bacterial host ranges were analyzed. The sources of these isolates have been compared, and the following indicate the percentage of samples that were positive for phage within each category: lysogenic phage, 54.5%, cosmetic samples, 22.7%, soil under hives, 18.8%, beehive materials, 15.9%, and other environmental sources, 14.8%. Host range data further indicated that each phage provided a unique pattern of infectivity on 11 P. larvae bacterial strains, with the exception of three pairs of identical patterns even when the phages were isolated from highly different sources. Two phages were capable of infecting all 11 strains and several phages were capable of a high degree of lytic activity on some strains. Sixteen phages were imaged by TEM with 13 belonging to the Siphoviridae. Isolated bacteriophages were combined to produce two different phage cocktails containing more or fewer phage types. These were administered to lab-reared honeybee larvae infected with cells and spores of P. larvae. Results of the lab-reared larval experiments indicate an increase in survival of infected larvae given phage compared to the survival of infected larvae without treatment. Both vegetative cells and spores resulted in death of the larvae with spores killing the larvae faster but with no significant difference by eight days between vegetative cells and spores of the same bacterial strain without phage treatment. The survival of larvae treated with the phage cocktail prior to infection increased by 70%, and was comparable with the survival rates of the phage cocktail controls. Collaboration with Dr. Todd Sandrin from Arizona State University resulted in a manuscript submission based on separation and rapid identification of Paenibacillus species using MALDI-TOF MS techniques. Objective 4: One field experiment was conducted near Bellingham, WA in summer, 2013. Improvement was seen in the hive that was highly infected with AFB, however the hive was not able to completely recover prior to the onset of winter. Removal of frames containing the worst infection and insertion of empty, new frames resulted in no visible return of disease over a two month period.

Publications

• Type: Conference Papers and Presentations Status: Other Year Published: 2013 Citation: Elekonich, M. M.; Abel-Santos, E.; Amy, P. S.; Wing, H.; Alvarado, I.; Yost, D.; and Smith, J. Microbial Biology as the Basis for New Treatments for American Foulbrood. 2013 Graduate & Professional Student Research Forum. March 16th, UNLV, Las Vegas, NV. Poster Presentation
• Type: Conference Papers and Presentations Status: Other Year Published: 2013 Citation: Jasmin C. Khilnani and Helen J. Wing. Testing Honeybee Antimicrobial Peptides to Counter the Growth of the Causative Agent of American Foulbrood Disease, Paenibacillus larvae. 52nd Annual Meeting of the Arizona/Southern Nevada Branch of the American Society for Microbiology. 13 April 2013, University of Arizona, Tucson, Arizona. Poster Presentation.
• Type: Conference Papers and Presentations Status: Other Year Published: 2013 Citation: Jasmin C. Khilnani and Helen J. Wing. Testing Honeybee Antimicrobial Peptides to Counter the Growth of the Causative Agent of American Foulbrood Disease, Paenibacillus larvae. 57th Annual Wind River Conference on Prokaryotic Biology. 5-9 June 2013, Estes Park, Colorado. Oral Presentation.

Progress 02/01/12 to 01/31/13

Outputs
Target Audience: We published data and made scientific presentations to reach basic science researchers in entomology and honey bee biology and in microbiology. We also did outreach to beekeepers and the general public. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided? Two PhD students and two MS students have been involved in the research along with 2 undergraduates. The two PhD students and one of the MS students all presented their research at conferences and within the department at the departmental graduate student colloquia. One MS student working with co-PD Dr. Abel-Santos and a co-author on the J. Bact. publication finished his MS degree. How have the results been disseminated to communities of interest? All members of the team have participated in outreach to beekeepers and to the public about honey bees and their diseases in general and this work in particular. See the products section for the scientific presentations at conferences and publications to disseminate the research to the scientific community. We have also received some coverage by the local news media: In the Las Vegas Review Journal: www.lvrj.com/news/unlv-researchers-fight-disease-affecting-honeybees-161759595.html The Las Vegas Weekly: http://www.lasvegasweekly.com/news/2012/jul/12/more-human-microbes-are-essential-part-our-worldan/ The Las Vegus Sun: http://www.lasvegassun.com/news/2012/jul/12/more-human-microbes-are-essential-part-our-worldan/ What do you plan to do during the next reporting period to accomplish the goals? Objective 1. Germinants and anti-germinants We plan to finish the tests of germinant and anti-germinant analogs on P. larvae and the tests of those analogs on uninfected and infected honey bee larvae to check for both toxicity and activity. We plan to pilot test experiments to identify the germinant receptors themselves, however full identification is beyond the scope of this project. Objective 2. Honey Bee Antimicrobial peptides. Currently, we are in the process of testing the antimicrobial activity of hymenoptaecin, defensin-1 and defensin-2 against P. larvae and other bacteria. Subsequently, we will combine the use of active AMPS to look for either additive or synergistic effects. In addition, the pro-peptide forms of each active AMP will be tested to determine their antibacterial activity. All of these assays test part i of Objective 2. As we move to determine the relative abundance of AMPs and pro-AMPs in the hemolymph of adults and 2nd instar larvae (part ii of objective 2) it will be important to know which forms of the peptides are antibacterial in nature. We expect to pursue these measures of the natural occurance of AMPs in larvae in the summer and fall when our available colonies are large enough and producing enough larvae to support the experiment. In the near future, we also plan to test the active AMPs and whole hemolymph from either honeybee larvae or adults (healthy and uninfected) against spores that are in the process of germinating (called spore outgrowth). We will test whether the AMPs affect this unique developmental stage of P. larvae. Objective 3. Phage lytic enzymes. Seventeen of the bacteriophages have been selected for DNA extraction, amplification, and restriction enzyme digest profiling. Thirteen of the phages are capable of lysing at least 80% of the 10 P. larvae strains on which they have been tested. These 13 phages are being considered as a phage cocktail for experimental treatments of infected bee larvae. Two of the phages were selected because they have the highest number of P. larvae strains that they completely lyse. One of these is also one of the phages that lyses at least 80% of the P. larvae bacterial strains. These two phages are being considered for lysin production. Three pairs of phage have similar or the same host range patterns and are being profiled to determine if similar phages were isolated from vastly different sources. Preliminary digest experiments using several different enzymes have narrowed down the number of digests needed for the profiles: SacI, BamHI, BglII, KpnI, and TasI. Objective 4. Testing use of products from Objectives 1-3. This objective will be the primary focus of year 3- testing exposure of larvae and adults to anti-germinants and mixtures of phage for bee safety in vitro and for efficacy on equipment and in hives. Our tests will begin with in vitro reared larvae and then move to adults/larvae in small nucleus colonies. We will also test the treatment of honey comb frames and hive tools following application of known amounts of spores. We are planning experiments torevisit the studies identifying infectious doses of spores needed to cause disease in honey bee larvae as part of the pilot data required to design experiments to test the efficacy of AMPs protection during P. larvae infection.

Impacts
What was accomplished under these goals? Objective 1. Germinants and Anti Germinants We found that P. larvae spores only germinated in response to L-tyrosine plus uric acid at physiologic pH and temperature. The germination profile of environmentally derived spores was identical to spores from a biochemically typed strain suggesting that the type strain remains indicative of the wild type bacteria. Because L-tyrosine and uric acid are the only required germinants in vitro, we screened amino acid and purine analogs for the ability to act as antagonists of P. larvae spore germination. Indole and phenol, the side chains of tyrosine and tryptophan, strongly inhibited P. larvae spore germination. Methylation of the N-1 (but not the C-3) position of indole eliminated the ability of indole to inhibit germination. These results were published in J. Bacteriology. We are continuing to screen additional chemically related compounds for germinant and antigerminant activity against P. larvae and B. subtilis as a control. We have begun testing the toxicity of these compounds to honey bee larvae raised in vitro. Objective 2. Honey Bee Antimicrobial peptides. In the past year, we have successfully developed two assays to test active honeybee antimicrobial peptides (AMPs) against Paenibacillus larvae; one assay was developed on semi-solid media and the other in liquid media. These assays allow us to gather both qualitative and quantitative data, respectively. There were some complications in the development process, but after testing different types of bacterial growth media, 3X R2B was found to prevent bacterial clumping and did not inhibit the action of the AMPs because it does not contain sodium chloride. To date, we have tested the following honeybee AMPs on P. larvae cultures with appropriate controls using these assays: apidaecin and abaecin. Importantly, our experiments directly test the effect of these AMPs on P. larvae, the causal agent of American Foulbrood Disease. This has not been tested before. At physiological concentrations (100 ug/ml; Casteels et al., 1989), apidaecin was not seen to affect the growth of either P. larvae or Bacillus subtilis (both gram-positive bacteria) in either assay, but a decrease in growth of P. larvae was observed at super-physiological concentration of 200 ug/ml. In contrast, the growth of the gram-negative bacterium Escherichia coli (MC4100) was affected by apidaecin supplied at concs. >1 ug/ml in liquid assays (100 times lower than concentrations found in the hemolymph of adult honeybees (Casteels et al., 1989)). These data confirm that apidaecin is more active against gram–negative than gram positive bacteria (Casteels et al., 1989). Based on our studies, we can now conclude that apidaecin alone is not an attractive candidate for AMP therapy/prophylaxis against American Foulbrood Disease. The AMP abaecin is present in adult hemolymph at 50 ug/ml (Casteels et al., 1990). In liquid cultures, concentrations of abaecin > 10 ug/ml and >1 ug/ml affected P. larvae and E. coli, respectively. Interestingly, with extended growth periods P. larvae incubated with effective concentrations of abaecin displayed higher optical densities. Although we are not sure what caused this effect, it is possible that an abaecin-resistant population was overgrowing the susceptible population in our assays. In contrast, abaecin did not affect the growth of any of the bacteria used in our semi-solid medium assays. It is fairly common for the activity of some AMPs to be diminished in semi-solid medium, so perhaps this observation is not too surprising. To conclude, since abaecin is active against P. larvae in our liquid assays, abaecin is an attractive candidate for AMP therapy/prophylaxis against American Foulbrood Disease. Objective 3. Phage lytic enzymes. In the past year we isolated additional phages from environmental samples sent from beekeepers in multiple states. Our total count is 31 as of March 7, 2013. Host range experiments using the soft agar overlay spot test method for Paenibacillus larvae bacteriophage have continued. We obtained two wild type strains of P. larvae from beehives afflicted by American Foulbrood disease and tested our 31 isolated phage for ability to lyse them. Additionally, we performed a host range of our bacteriophage on a species of Paenibacillus other than P. larvae and on genera of bacteria other than Paenibacillus with the purpose of evaluating the host specificity of the phage. To date, the twelve bacterial species from four other genera are not remotely susceptible to P. larvae bacteriophage.The other Paenibacillus species, was very slightly susceptible to six of the 31 bacteriophage.These results indicate the high host specificity in the phage and that using them in a hive wouldunlikely to affect non-AFB causing bacteria. Objective 4. Testing use of products from Objectives 1-3. Nothing to report- we will begin this objective in year 3.

Publications

• Type: Journal Articles Status: Published Year Published: 2013 Citation: Alvarado,I.; Phui, A.;. Elekonich, M.M.; Abel-Santos, E (2013) Requirements for in vitro germination of Paenibacillus larvae spores, Journal of Bacteriology, 195 (5), 1005-1011. doi:10.1128/JB.01958-12
• Type: Conference Papers and Presentations Status: Other Year Published: 2012 Citation: Yost, D. and P.S. Amy. Sources of Bacteriophages which Infect Paenibacillus larvae, an Important Honeybee Pathogen. Abstract submitted and accepted for the American Society for Microbiology annual meeting in San Francisco, CA. Poster presentation June 2012.
• Type: Conference Papers and Presentations Status: Other Year Published: 2012 Citation: Developing a Method to Test Honeybee Antimicrobial Peptides Against Paenibacillus larvae Jasmin Smith & Helen J. Wing. AZ/Southern Nevada branch of ASM. ASU, Tempe, AZ (April 21st 2012)
• Type: Conference Papers and Presentations Status: Other Year Published: 2012 Citation: Developing a Method to Test Honeybee Antimicrobial Peptides Against Paenibacillus larvae Jasmin Smith & Helen J. Wing. 56th Annual Wind River Prokaryotic Conference (June 6-10, 2012). Mt. Charleston, Nevada.
• Type: Conference Papers and Presentations Status: Other Year Published: 2013 Citation: Microbial Biology as the Basis for New Treatments for American Foulbrood. 1st Annual STEM Conference (January 14th 2013) UNLV, Las Vegas, NV.
• Type: Conference Papers and Presentations Status: Other Year Published: 2012 Citation: Elekonich, M.M., Abel-Santos E., Amy P. S., Wing H., Alvarado I., Yost D., and Smith J. (2012, Nov.) Microbial Biology as the Basis for New Treatments for American Foulbrood, USDA/AFRI Insects and Nematodes Program Project Director Workshop; Oak Ridge, TN.

Progress 02/01/11 to 01/31/12

Outputs
OUTPUTS: Objective 1. Germinants and Anti Germinants We purchased P. larvae larvae 9545 and P.larvae pulvifaciens 49843 strains from the American Tissue Culture Collection, refined methods to produce a spore suspension and identified the germinants and anti-germinants. Objective 2. Honey Bee Antimicrobial peptides. As of August we recruited Jasmin Smith who became a MS student(Spring 2012). Jasmin has been learning how to culture P. larvae, how to recognize when cultures are contaminated and to conduct regular checks to verify the purity of the strain. Recently, Jasmin has been using 96 well plates to grow cultures of P. larvae so we can reduce the amount of peptides used during the antimicrobial peptides resistance /susceptibility assays. This has not been trivial as P. larvae cells appear to aggregate under usual growth conditions, leading to well-to-well inconsistencies. We are experimenting with different kinds of media to see if this abolishes cell aggregation. Once this has been resolved, we will first test the antimicrobial peptide apidaecin, the shortest peptide, with suggested bacteriostatic function. Objective 3. Phage lytic enzymes. P. larvae bacteriophage were isolated using an overnight P. larvae NRRL 2605 host cell suspension supplemented with potential phage samples from various sources, e.g., 10 g soil, 2-3 g wax, etc. Overnight growth of the mixture was followed by standard centrifugation and filtration of the supernatant. The filtered supernatant was then reinoculated with 1.0 ml P. larvae fresh broth culture and incubated by shaking at 100 rpm in a 37C environmental shaker. The centrifugation and filtration steps were repeated the next day to complete the phage lysate. This lysate was then tested for bacteriophage using a soft agar overlay and P. larvae 2605 as the host cell. Lysates could be amplified to increase the phage titer by another round of growth, centrifugation and filtration. Objective 4. Testing use of products from Objectives 1-3. Student Israel Alvarado is learning to rear larvae in vitro to test the germinants and anti-germinants in the coming months. PARTICIPANTS: Michelle Elekonich, Project Director, coordinated all aspects of the project and co-mentored Israel Alvarado, consulted with other members on aspects of bee biology. Penny Amy, PI, lead for the phage research; mentor for Ms. Yost. Ernesto Abel-Santos, PI, lead for the germinant-antigerminant work, co mentor for Mr. Alvarado. Helen Wing, PI, lead for the antimicrobial protein research and mentor for Jasmin Smith. Israel Alvarado, graduate student, germinant and antigerminant work, in vitro larval rearing Diane Yost, graduate student, phage research Jasmin Smith, graduate student, antibacterial protein research The team of 4 PIs and 3 graduate students includes 2 Hispanic men and 5 women: 4 Caucasian; one African American. TARGET AUDIENCES: Outreach: Outreach to bee keepers in PA, NV and IA describing the problem of AFB and our approach resulted in environmental samples of wax, soil, bees, propolis, royal jelly, and one scale sample from an infected hive. Israel Alvarado from the Elekonich and Abel-Santos labs, presented a talk on crop pollination and bee diseases to 3rd graders at Tartan Elementary School in North Las Vegas. He also presented at the 5th Annual Wind River Conference on Prokaryotic Biology, Estes Park, CO. Dr. Wing described our work during an invited seminar in the College of Life Sciences at Brigham Young University in March 2011. This college is an active participant in the "Phage Hunters Program" sponsored by Howard Hughes Medical Institute, and our phage work generated considerable interest. Dr. Elekonich described our work in an invited seminar at Georgetown University and in a talk at the National Science Foundation. PROJECT MODIFICATIONS: Not relevant to this project.

Impacts
We have modified traditional techniques to culture, maintain and create spores of P. larvae. To grow P. larvae in overnight cultures we use Brain Heart Infusion broth supplemented with 1 mM CaCl2 and MgCl2 inoculated using a 1.5 ml freezer stock and shaken at 37C overnight or until desired turbidity. Even under these optimal conditions, the maximum OD 600nm obtained is 1.0. If growth occurs in broth culture it may be visible three or four days after the culture is started. Broth cultures older than 48 hrs, and BHI agar plates older than 4 days were not viable. Additionally, P.larvae cultures often die after they are transferred to a fresh medium. We maintain stock cultures on agar using R2A (Difco) agar plates prepared by streak-plating from overnight broth cultures followed by incubation for 24-48 hrs at 37C. Only the lower nutrient medium (R2A) maintained plate cultures for 1-2 weeks. Previous research suggests P. larvae forms spores in liquid, solid, or solid-liquid media. We found that P. larvae larvae 9545 strain was unable to undergo sporulation by any method. P. larvae pulvifaciens 49843 produced spores readily on agar plates incubated for 7 days at 37C in a CO2 incubator. Objective 1. P. larvae spores germinated rapidly in response to L-tyrosine and uric acid, neither compound alone was sufficient. Compounds with similar chemical structure to P. larvae germinants did not stimulate germination. Germination was slightly inhibited by purines and unaffected by D-tyrosine. Indole and phenol, the degradation products of tryptophan and tyrosine, strongly inhibited spore germination. Strikingly, spore germination was not observed in complex media or in an artificial medium containing universal germinants. Objective 3. During an environmental survey for bacteriophage capable of infecting P. larvae we obtained ~100 samples from soil, bee hives, honey, flowers, propolis, wax, dead bees, royal jelly, and cosmetic products containing beeswax. The samples were screened by a series of washing, amplification, filtration and testing steps that gave rise to 31 phages from various sources. Prior to this study, the only isolated phages came from lysogenic genomes incorporated into the chromosomes of various P. larvae strains. Each new phage was plated on the host strain, NRRC 2605, with a soft agar overlay method. Plaques were picked and enriched by adding putative phage to broth containing the host strain 2605 and incubating the mixture overnight with shaking. Isolated phage were tested on seven additional P. larvae strains to determine the host ranges. On average, the phages infect 3.3 out of 8 strains. One phage, however, is capable of infecting all 8 strains tested. We developed a spot test for conducting host range experiments because many of the phages only produce minute, turbid plaques. Currently, the Amy lab is working on finding an effective method to purify DNA from the isolated phages so we can assess how many unique phage types are in our collection using DNA restriction patterns. The phages with the highest clearing in their plaques and the broadest host ranges are candidates for lysin gene isolation.

Publications

• Yost, D. and P.S. Amy. Sources of Bacteriophages which Infect Paenibacillus larvae, an Important Honeybee Pathogen. Abstract submitted to the American Society for Microbiology annual meeting in San Francisco, June, 2012.
• Alvarado, I.; Phui, A.; Elekonich, M.M.; Abel-Santos., E. (2011, June) American foulbrood: germination of Paenibacillus larvae in complex media; 55th Annual Wind River Conference on Prokaryotic Biology, Estes Park, CO.
• A manuscript is being written titled: Requirements for in vitro germination of Paenibacillus larvae spores by Israel Alvarado, Andy Phui, Ernesto Abel-Santos, and Michelle M. Elekonich to be submitted in 2012. The paper will be the first publication about P. larvae spore germination which is the initial step in AFB disease.A manuscript is being written entitled: Sources and Characterization of Bacteriophage that Infect Paenibacillus larvae which will also be submitted in 2012.