Dr. Kirby C. Stafford IIIDepartment of EntomologyThe Connecticut Agricultural Experiment StationNew Haven, CT 06504Evaluation of Natural Products for the Control of the Tick Vectors of LymeDisease SpirochetesProject Abstract The objective of this proposed project is to identify and devise formulations ofnatural products, particularly the eremophilane sesquiterpene nootkatone, andevaluate their repellency to and effectiveness for the reduction of questingnymphal Ixodes scapularis in the field. We propose several candidate naturallow-toxicity chemicals potentially toxic or repellent against I. scapularis. Workingwith a scientist with the USDA Agricultural Research Service, we will improve thecurrent emulsifiable concentrate (EC) formulation of nootkatone and developpreliminary encapsulation techniques for this chemical using coacervation,Fantesk encapsulation, and spray dried encapsulation. Formulation samples willbe screened in the laboratory at The Connecticut Agricultural Experiment Stationfor efficacy against I. scapularis and the formulations will be optimized foraqueous spray applications. Field trials will be conducted in communities inFairfield and Litchfield Counties of Connecticut; areas with high tick densities andendemic for Lyme disease. The repellency of the new natural product candidateswill be evaluated initially in the laboratory against I. scapularis. In the field,repellency of nootkatone, and other candidate natural products will be evaluatedusing treated and untreated flannel tick drags. In addition, we will bring severaladded components to the proposed project beyond the stated objectives in thefunding opportunity announcement that will enhance the outputs and outcomesof the research. Chemical analysis of field samples will provide nootkatoneresidue-degradation data. In addition, we propose to further test the compatibilityand field efficacy of the entomopathogenic fungus Metarhizium anisopliae Strain52 with nootkatone and other natural products, which may permit effective use oflower concentrations of the natural compounds in the field for a more economicaland integrated natural approach to tick control. PI: Dr. Kirby C. Stafford, IIIVice Director, Chief Entomologist, State EntomologistDepartment of EntomologyThe Connecticut Agricultural Experiment Station123 Huntington Street, P.O. Box 1106,New Haven, CT 06504Tel. (203) 974-8485Fax (203) 974-8502E-mail: Kirby.Stafford@po.state.ct.usPROJECT NARRATIVEEvaluation of Natural Products for the Control of the Tick Vectors of LymeDisease SpirochetesA.
SPECIFIC AIMS The aims of this proposed project are to identify and devise formulations ofnatural products, particularly the eremophilane sesquiterpene nootkatone, andevaluate their effectiveness for the reduction of questing nymphal Ixodesscapularis in the field in areas highly endemic for Lyme disease. This project willassist in developing new technology for the management of tick vectors of Lymedisease spirochetes, determine how to integrate natural botanical compoundswith a biological control agent, and ultimately provide an alternative tickmanagement option to conventional acaricides for individual stakeholders andthe land care industry. Within the project goal to evaluate the efficacy of naturalproducts for the control of the tick vectors of Lyme disease spirochetes, there arefive research objectives. We propose a study on the control of blacklegged ticksusing natural products as outlined in the funding opportunity announcement,including but not limited to nootkatone, repellency of these natural products to I.scapularis, and, in addition, test the compatibility and field efficacy of theentomopathogenic fungus Metarhizium anisopliae Strain 52 with nootkatone andother natural products for a more integrated natural and biological approach.People prefer the use of natural compounds or biological agents for pest controland personal protection measures. Availability of natural alternative controlmaterials and agents may lead to increased acceptance of applications ofacaricidal agents to the landscape and increased tick control. The objectives are:Objective 1. Identify all-natural, low-toxicity chemicals extracted from botanicalsources, including the eremophilane sesquiterpene, nootkatone, toxic to thenymphal stage of the blacklegged tick, I. scapularis, and devise formulations(water miscible, emulsifiable, and microencapsulated or other slow-releaseformulations to extend residual activity) for aqueous application to vegetation andleaf litter for the control of host-seeking I. scapularis (FOA research objectives 1& 2).Objective 2. Field test formulations against populations (June peak >0.1/m2) ofthe blacklegged tick in Lyme disease endemic areas and determine whethercontrol of ticks using test chemicals is accomplished by direct toxicity to ticks infield plots via approach outlined in the proposal announcement (FOA researchobjectives 3 & 4).Objective 3. Test repellency of the natural products against I. scapularis in thelaboratory and in the field with treated and untreated flannel tick drags (FOAresearch objective 5).Objective 4. Test formulations of natural products, particularly nootkatone, inthe laboratory and field in combination with the entomopathogenic fungus, M.anisopliae Strain 52, for the control of I. scapularis nymphs. These objectives can be broadly divided into product development and fieldevaluation of natural products for repellency and control of the tick. The field trialswill be conducted in communities in Fairfield and Litchfield Counties ofConnecticut; areas highly endemic for Lyme disease. We will bring several addedcomponents to the proposed project that will enhance the outputs and outcomesof the research. In cooperation with the Department of Analytical Chemistry atThe Connecticut Agricultural Experiment Station (CAES), we will providequantitative assessment of residual nootkatone after application in the field.Working with a scientist with the USDA Agricultural Research Service, slowrelease formulation(s) to extend nootkatone efficacy will be developed andtested. Preliminary tests show that germination of M. anisopliae is not affected by0.1% nootkatone. Therefore, M. anisopliae will also be incorporated into the testof natural product formulations to determine if the agents can be integrated into acontrol program and permit effective use of lower, economical concentrations ofthe natural compounds. Nootkatone will be obtained from commercial sourcesand Novozymes Biologicals, Inc. (Salem, VA) will provide the M. anisopliae.B. BACKGROUND AND SIGNIFICANCE Lyme disease (LD) is the most important tick-borne disease in the UnitedStates. A record 23,763 human cases were reported in 2002, closely followed by23,305 in 2005 (Centers for Disease Control and Prevention 2003; Centers forDisease Control and Prevention 2007), which probably represent only about 10-20% of diagnosed cases. The disease is caused by the bacterium, Borreliaburgdorferi Johnson, Schmid, Hyde, Steigerwalt & Brenner, which is transmittedby the bite of the blacklegged tick, I. scapularis Say. There is a strong correlationbetween the abundance of infected I. scapularis, specifically the nymphal stagethat is most abundant in spring and summer months (Mather et al. 1996; Staffordet al. 1998), and the incidence of LD. Furthermore, this vector is responsible fortransmitting the agents of human babesiosis and human granulocyticanaplasmosis, two emerging tick-borne infections in the US. Connecticut has thehighest or among the highest rates for LD in the United States, with a rate of 136cases reported per 100,000 persons in 2002 (4,631 reported cases) and 53cases per 100,000 population in 2005 (1,810) (Ertel and Nelson 2003; Ertel et al.2006). The drop in cases from 2003 is largely because of changes in reporting.Fairfield County has the greatest number of cases, generally accounting forabout a third of Connecticut's total and Litchfield County has the highest rate ofLD (299 per 100,000 population). In addition, there were 49 confirmed and 154probable cases of anaplasmosis and 69 confirmed cases of human babesiosis inthe state in 2002 (Ertel et al. 2003; Ertel et al. 2003). In the northeastern states, individuals with the highest risk for tick bites andLyme disease are those residing in suburban residential developments withadjacent wooded tracts, and those with rural homes in a woodland environment,where hosts for the tick flourish (Falco and Fish 1988; Falco and Fish 1988;Stafford and Magnarelli 1993). Cases occur most commonly in children aged 5-14 years and those 45-54 years, which probably reflects outdoor activity in thesummer months when nymphal ticks are active. Most cases of LD appear to beacquired peridomestically. Therefore, control of ticks in residential locations,particularly in areas of high disease incidence, are expected to reduce thenumber of local LD cases. A variety of vector control approaches have been explored to determine theirefficacy in reducing tick abundance (Stafford and Kitron 2002; Ginsberg andStafford III 2005). These include personal protective measures, hostmanagement, habitat modification, acaricide applications, host-targetedacaricides, and biological control. While most of these approaches have met withmixed success and acaricides can provide excellent tick control over relativelylarge areas, environmental concerns have restricted their acceptance and broaduse. The acaricidal treatment of important hosts, such as white-footed mice andwhite-tailed deer also has a substantially reduced environmental impact and hasalso been shown to reduce tick abundance. Recently completed trials of therodent bait box in southwestern Connecticut by CAES have shown reduced tickdensities at residential home sites though little impact was observed at homes inmore rural northwestern parts of the state. However, the commercially developedrodent bait box containing fipronil is no longer being manufactured due to costissues. The 4-poster technology for the passive application of an acaricide todeer, though providing significant tick control (Pound et al. 2000; Carroll et al.2002; Carroll and Kramer 2003; Solberg et al. 2003), has not been widelyadopted in the northeast for control of the blacklegged tick amid cost issues,placement restrictions, and concerns by wildlife agencies of feeding the corn baitto deer and emerging chronic wasting disease of deer. Acceptance of chemical acaricides in some communities is low. Forinstance, a study of residents in the towns of Westport and Weston, CT in whichLyme disease attitudes and behaviors were surveyed (under CDC cooperativeagreements to the Connecticut Department of Public Health and CAES) foundthat only 22.5% of residents reported having sprayed a chemical pesticide for tickcontrol, and 69.5% indicated that the use of pesticides was not very likely orlikely at all. Several years of community education on how to target and minimizepesticide use has increased use of acaricides to 38% of survey respondents (CTDPH, unpublished data). Yet clearly, there is a strong niche for alternative controlmethods or compounds and new tools are needed. Biological agents such asentomopathogenic nematodes and parasitoid wasps have had limited success inreducing numbers of I. scapularis. Engorged I. scapularis females aresusceptible to fatal infection by steinernematid nematodes, but unfed femalesand younger (immature) stages are not. Furthermore, sensitivity of nematodes tolow fall season temperatures in the northeastern US limits their potentialapplication for control of this tick at present. Likewise, the blacklegged tick isparasitized by the chalcid wasp, Ixodiphagus hookeri, but only at high tick andhost deer densities. Thus, it is unlikely that a control approach using this waspspecies will be successful (Stafford et al. 2003). By contrast, natural productsand entomopathogenic fungi could provide safer, organically acceptable tickcontrol. A combination of these strategies may provide the least toxic options for tickcontrol in the residential landscape. People prefer the use of natural compoundsor biological agents for pest control and personal protection measures. Publicinterest in safer alternatives is reflected in a growing interest in and accreditationof landscape professionals in organic land care and the use and acceptance ofnatural, organic, and herbal products have been on the rise in recent years(Tanneeru 2006; Cunningham 2007). Repellent products containing natural,botanical ingredients have increasingly become available to the public. Botanicalcompounds have long been known to have repellent and/or toxic propertiesagainst arthropods. Until recently, information on the activity of natural productsagainst ticks has been relatively limited. Tick products currently are restricted tothose containing d-limonene for flea and tick control on pets, which has littleefficacy against I. scapularis (Panella et al. 1997). Not all botanical extracts orcompounds are effective against arthropod vectors or may offer some protectionagainst mosquitoes, but not ticks (e.g. citronella). Many botanicals offer only ashort duration of protection (3 to 30 minutes), a problem frequently due to highvolatility of the compound requiring frequent replacement. Some African plantshave strong tick-repellency (Kaaya 2000). USDA-ARS scientists have shown thattwo American beautyberry, Callicarpa americana, compounds, callicarpenal andintermedeiol, may be effective repellents against I. scapularis (Pons 2007). Theactive ingredient of extracts of lemon eucalyptus oil, citriodiol, has been shown ina few studies to provide some repellency and protection against tick bite (Gardulfet al. 2004; Garboui and Jaenson 2006; Jaenson et al. 2006), all against I.ricinus, the tick vector for Lyme disease in Europe. There does not appear to beany peer-reviewed studies evaluating the repellency of lemon-eucalyptus againstI. scapularis. The extracts or oils of several other plants have also been found tohave repellent properties against I. ricinus (Jaenson et al. 2005). The essentialoil from the heartwood of Alaska yellow cedar, Chamaecyparis nootkatensis, wasfound to be a highly effective toxicant against nymphal I. scapularis (Panella etal. 1997). Various components of the essential oil, especially nootkatone, aneremophilane sesquiterpene, were subsequently shown to have 10x greateractivity than the oil itself (Panella et al. 2005). However, a 1-2% rate appearsnecessary for control in the field (Marc Dolan, personal communication), a ratefar above the LC50 of 0.0029% (Panella et al. 2005). The nootkatone derivedfrom grapefruit oil and three other natural compounds were found to be highlyrepellent, comparable to DEET, in a screening assay against I. scapularis(Diethrich et al. 2006). Clearly, there is a large disparity in efficacy of botanicalcompounds against ticks, ranging from highly effective to no observable effect asa repellent or toxicant. The recognized safety of some compounds (e.g.;citronella and citronella oil, eugenol, garlic and garlic oil, geraniol, geranium oil,lemongrass oil, rosemary and rosemary oil, thyme and thyme oil) by the EPA (40CFR 152.25 - minimum risk pesticides) and their exemption from therequirements of the Federal Insecticide, Fungicide, and Rodenticide Act (FIFRA)has lead to their use in formulated products of unknown effectiveness againstticks. While no claim to control disease may be made, a few products do claim tocontrol 'deer ticks'. Active ingredients approved by the Organic Materials ReviewInstitute (OMRI) include neem products, Bacillus thuringiensis (BT), Beauveriabassiana (fungus), garlic, pyrethrin, rosemary oil, and insecticidal soap.Clarification of the efficacy of some compounds and the development of newones are badly needed. A number of fungi have been isolated from ticks, primarily from engorgedfemales collected off deer, although some fungal species have been found onlarvae, nymphs, and unfed females. These include Paecilomyces farinosus,Lecanicillium (formerly Verticillium) lecanii, Beauveria bassiana, and some otherspecies from these three genera in the Hyphomycetes family. Recovery ratesare low, however, generally less than 5%. Lecanicillium lecanii appears to be acommon fungal pathogen of I. scapularis and I. ricinus in nature.Entomopathogenic fungi such as Beauveria bassiana and Metarhiziumanisopliae have been reported to cause mortality in I. scapularis. Two products,BotaniGard (Mycotech, Butte Montana) and Naturalis T & O (Troy Biosciences,Phoenix, Arizona) provided effective tick control in the laboratory and in fieldtrials (S. Allan and K. Stafford, unpublished data), but are not labeled for tick use.Initial field trials in 2002 with the entomopathogenic fungus (Metarhiziumanisopliae Strain 52) provided high levels of control (80-85%, K. Stafford,unpublished data). This fungus was provisionally registered by the EnvironmentalProtection Agency (EPA) for tick biocontrol. Metarhizium is a naturally occurringsoil fungus that is considered nonpathogenic to mammals. This fungus possesminimal risk to non-target organisms and does not harm many beneficial insectssuch as honey bees, green lacewings, lady beetles, parasitic Hymenoptera orearthworms at rates used. Field applications with M. anisopliae Strain 52 (Tick-Ex', Novozymes Biologicals, Inc., Salem, VA) in 2007 resulted in around 90%control for several weeks after a second application and overall seasonal controlof 77.8% at the higher rate tested (see preliminary studies/progress report). Fullregistration is now pending. It was clear however that applications must be madeduring nymphal tick activity. Nootkatone has excellent knock-down propertiesand the fungus could compliment the natural compound to provide improved tickcontrol. However, nootkatone appears to lack a long lasting effect in the field. Formulation technology can enhance the activity of many pesticideapplications by extending storage stability, improving dispersion in the spraytank, and extending residual activity after application. Nootkatone has twoadverse characteristics that may specifically be addressed by proper formulationdevelopment, and specifically by encapsulation. First, the nootkatone is insolublein water (Material Safety Data Sheet, Bedoukian Research, Danbury, CT).Second, applications of nootkatone have demonstrated a short period of residualactivity (M. Dolan, personal communication). Specific reasons for contributing tothe rapid loss of efficacy are not known, but likely are related to loss by volatilityor adsorption into the treated substrate (plants, leaf litter, soil, etc.). These lossesmay be reduced by encapsulation formulations that provide a barrier betweennootkatone and the air and/or treated substrate. There is high probability ofextending residual activity of nootkatone by the application of the appropriateformulation technology. Many encapsulation techniques including spray drying,polymer encapsulation and coacervation are being researched, often for use inthe food industry (Gouin 2004), and these techniques may be suitable forencapsulation of nootkatone. Likewise, when considering integrated applicationswith entomopathogenic fungi, encapsulating nootkatone may again provide abarrier that will allow mixing higher concentrations of nootkatone with spores forapplication without inhibiting spore germination. At this time, nootkatone isexpensive. If a lower rate of nootkatone acts in combination or synergisticallywith the fungus, then costs could be reduced. An encapsulated formulation maybenefit applications of nootkatone by extending residual activity or allow flexibilityto tank mix with fungal agents without detrimental affects on spore germination.Natural products alone or in combination with a Metarhizium formulation couldalso potentially be used in wild animal host-targeted applications. A natural, integrated tick management program will require a variety of toolsthat can be used under different circumstances and with different segments ofthe human population. We propose to develop, test, and refine natural products,especially nootkatone, as well as examine their compatible use with the fungusM. anisopliae (Tick-Ex) against the nymphal stage of the blacklegged tick, I.scapularis.C. PRELIMINARY STUDIES/PROGRESS REPORT Scientists at the Connecticut Agricultural Experiment Station (CAES) havebeen involved in studies of tick ecology and tick control technologies for 20years. Some tick control trials and testing of new technology is ongoing orrecently completed and has not yet been published. Other specialists in relatedfields chemistry, mycology, and wildlife biology, are also on staff and available forcollaboration or consultation. The principal investigator has a supervisorypesticide application license and the technician on the project has a pesticideapplication license. Strategies that have been evaluated for tick control, most of which haveinvolved CAES, include habitat modification, burning vegetation (Mather et al.1993; Stafford 1998), the use of parasitoid wasps that infect I. scapularis(Stafford et al. 1996; Stafford et al. 2003), chemical acaricides (Stafford 1991;Solberg et al. 1992; Curran et al. 1993; Schulze et al. 2000; Schulze et al. 2001),pesticide treatment of rodents and deer (Pound et al. 2000; Rand et al. 2000;Solberg et al. 2003; Dolan et al. 2004), exclusion of deer from defined areas(Daniels et al. 1993; Stafford 1993; Daniels and Fish 1995), and the reduction ofvertebrate host numbers (Wilson et al. 1984; Deblinger et al. 1993; Stafford et al.2003). The PI and CAES staff has extensive experience in conducting andevaluating tick control trials with natural (e.g., pyrethrin) and synthetic acaricidesand with entomopathogenic fungi. The application of entomopathogenic fungi totick habitat or vertebrate hosts to control ticks is a more environmentallyacceptable approach than chemical use, and fungi are one of the more promisingtick biological control agents that could provide a natural complement to botanicalcompounds. Initial field trials of a new commercial strain of M. anisopliae in 2002provided 80-85% control of nymphal I. scapularis (K. Stafford and S. Allan,unpublished data) and led the Environmental Protection Agency (EPA) toprovisionally register this product for the control of ticks (Tick-Ex') and otherpests. We have continued these evaluations in the laboratory and again in thefield in 2007 as product became available. Novozymes Biologicals, Inc. acquired Earth BioSciences, Inc. on September30, 2006, including the Metarhizium anisopliae insecticide product line. Theacquisition according to the press release fits into Novozymes position as aleader in the research, development, and manufacture of biotechnology productsand natural pest technologies. The company has made the additionalinvestments necessary to begin production and permit expanded field trials in2007. Additional studies were needed to examine field efficacy of the emulsifiableconcentrate (EC) formulation and timing of application for full registration andintegration into tick management program. Field trials to evaluate efficacy andspore longevity in the field were conducted by The Connecticut AgriculturalExperiment Station in summer 2007, May through August in northwesternConnecticut (CT). Trials were conducted at lawn edges at home sites in the threetowns of Salisbury, Falls Village, and Cornwall in cooperation with the TorringtonArea Health District. Treatments plots were established at the perimeters of theproperties in tick habitat ranging from 39-116 m2 at home sites (Table 1). The oil-based formulation (Tick-EXTM EC, Novozymes Biologicals, Inc., Salem, Virgina)with a concentration of 3.9x109 cfu/ml was sprayed by a commercial applicator(NaturaLawn of America, Danbury, CT) as directed by the principal investigatorsusing a high volume hydraulic sprayer at two application rates (2.6 fl oz /1000 ft2'lower rate' and 10.4 fl oz/1000 ft2 'higher rate') at 150 psi. A total of 20 homesites were sprayed - 11 sites were treated with lower rate (3.2x105 cfu/cm2 ) and9 sites were treated with five times higher rate (1.3x106 cfu/cm2). Based on thenumber of nymphs/100m2 recovered from these sites from 2003-2006, therewere no significant differences in nymphal abundance between the sites in thetreatment blocks. Control sites (n = 21) received no treatment. The fungus wasapplied two times; once May 8th and 9th and again on June 29th and July 2nd. Theeffective rates (viable spores applied) were 2.0x105 and 8.2x105 cfu/cm2 for lowand high rates, respectively, for the 1st application and 2.2x105 and 9.0x105cfu/cm2 for the low and high rates,respectively, for the 2nd application. After thefirst application 39.8 and 9.9 % control was observed from the lower and higherrate treated sites, respectively for the approximately 5 weeks post application.The percent controls obtained after the second application from lower and higherrate treated sites were 53.2 and 73.8%, respectively, for the period ending July30, 2007 and 36.5 and 77.8% for the 8 weeks ending August 20, 2007. However,percent control for the 3 weeks following the 2nd application was 87.1 and 96.1for the low and high rates, respectively. There was a significant difference in thetreatment groups in the number of nymphs collected for the whole samplingseason (DF = 2, F = 5.271, P = 0.005). In pair wise multiple comparisonprocedures, there was a highly significant difference between the nymphscollected from control and lower rate sites (t = 2.888, P = 0.004) as well asbetween the control and high rate sites (t = 2.319, P = 0.021). No significantdifference was observed between the low and high rate sites. Preliminary tests at CAES show that germination of the fungus is not affectedby 0.1% nootkatone, but there is inhibition at 1.0% (Bharadwaj and Stafford,unpublished data). As noted previously, a 1-2% rate of nootkatone appearsnecessary for control in the field (Marc Dolan, personal communication), a ratefar above the LC50 of 0.0029% (Panella et al. 2005). It may be possible to use alower economical rate of nootkatone when combined with M. anisopliae. In the course of the tick control research over the past 20 years, extensivecontacts have been developed with the Connecticut Department of Public Health,many local and regional health districts, people and businesses, such ascommercial applicators, and many stakeholders, such as homeowners, many ofwhom have provided access to their properties for tick surveys and controlstudies over many years. There is strong interest by the public in the continuationof these studies. The health departments will continue to work with us (see lettersof support). The principal investigator has provided information on ticks and tickmanagement to stakeholders through fact sheets, talks, lectures, workshops, andmost recently, a Tick Management Handbook (see Outreach below).D. RESEARCH DESIGN AND METHODSObjective 1. Identify all-natural, low-toxicity chemicals extracted from botanicalsources, including the eremophilane sesquiterpene, nootkatone, toxic to thenymphal stage of the blacklegged tick, I. scapularis, and devise formulations(water miscible, emulsifiable, and microencapsulated or other slow-releaseformulations to extend residual activity) for aqueous application to vegetation andleaf litter for the control of host-seeking I. scapularis (FOA research objectives 1& 2). Natural products evaluated against I. scapularis must include theeremophilane sesquiterpene, nootkatone, but need not be limited to thischemical. Other possible natural compounds for repellent and toxicity testinginclude nepetalactone (from catmint, Nepeta cataria) and curcumin with mustardoil (from turmeric, Curcuma longa, occasionally used as a tick repellent in India).Carvacrol, another component of the Alaska yellow cedar extract, has beenshown to have significant biological activity against I. scapularis, but has notbeen tested in the field. Some companies are using botanical or natural materialsfrom the EPA's 25b minimum risk pesticide list in mosquito and tick controlproducts. Products containing these active ingredients are exempt from therequirements of the Federal Insecticide, Fungicide, and Rodenticide Act (FIFRA).While products cannot bear claims to control disease or arthropods carryingspecific diseases, including ticks that carry Lyme disease, they are labeled forticks. Garlic-based products (e.g. Mosquito Barrier and Tick and Flea Solution)are already on the market and being applied for the control of I. scapularis.However, no data on its efficacy is available. The CDC project scientist willparticipate in the selection and approval of test natural acaricides. We will devise water miscible, emulsifiable formulations for testing the naturalproducts. The current emulsifiable formulation of nootkatone uses d-limoneneand EZ-Mulse (non-ionic surfactant for use with citrus terpenes) (M. Dolan,personal communication), but does not appear to possess sufficient residualactivity. Absorption by substrate, volatility, and degradation by environmentalfactors are common problems with a natural material. Dr. Robert Behle, NationalCenter Agricultural Utilization Research, USDA Agricultural Research Service,Peoria, Illinois, will evaluate the current EC for emulsification characteristics(ASTM standard E 1116) and determine if improvements are necessary toestablish a standard EC formulation to compare with subsequent formulationsamples. Slow/extended release or encapsulated formulation(s) to extendefficacy will be developed and evaluated in the laboratory for field testing in year2 and 3 of the project. We propose three techniques for encapsulatingnootkatone. These are: coacervation, an established technique in industry thatuses dilute solutions of carbohydrate and proteins to form a coating that can becured by crosslinking the shell material (Yeo et al. 2005), the FaneskTM process,developed in the USDA-ARS laboratory, that uses starch and a steam jet toencapsulate oils (Fanta et al. 1999) and spray dryer encapsulation using apolymer to encapsulate the oil emulsion in aqueous phase, then flash drying toform a dry powder (Gouin 2004). Storage stability of formulations for physicalcharacteristics, mixing characteristics, and loss of active agent at quarterly timeintervals for up to one year will also be examined and stored samples will beprovided for efficacy evaluation against I. scapularis. Efficacy Testing of Nootkatone Formulations - The extended releaseformulations will be evaluated in comparison with emulsifiable formulationsagainst I. scapularis nymphs in the laboratory using a modification of thedisposable pipette method (Barnard et al. 1981; Maupin and Piesman 1994).Nymphs will be exposed to inner surface of the pipette treated with the naturalproduct and extended formulation(s) for 24-h and observed for mortality. LC50and LC90 will be calculated using probit analysis. Suitable formulations will becandidates for semi-field and field trials. Nymphs of I. scapularis for thebioassays will be obtained by rearing female ticks collected in the field on NewZealand White rabbits in our tick rearing facility at Lockwood Farm, Hamden,Connecticut, as approved by the Experiment Station's Animal Care and UseCommittee (IACUC) (Protocol S01-05). Larvae hatching from the eggs laid by thefemale will be fed on laboratory mice to obtain the nymphs used in the trials.Objective 2. Field test formulations against populations (June peak >0.1/m2) ofthe blacklegged tick in Lyme disease endemic areas and determine whethercontrol of ticks using test chemicals is accomplished by direct toxicity to ticks infield plots via approach outlined in the proposal announcement. Based on laboratory results, we propose to evaluate selected naturalproducts in the field for acaricidal activity, repellency (see Objective 3), andresidual activity against nymphal I. scapularis, particularly encapsulatedformulations of nootkatone. Field Applications - The field applications will be conducted in woodlandtracts on residential property in southwestern (Fairfield County) and northwestern(Litchfield County) Connecticut, areas highly endemic for Lyme disease. Ticksare abundant and densities easily may exceed 0.1 per square meter at untreatedproperties. Residential properties currently participating in current tick samplingand tick testing programs in communities in both counties will be recruited for theproposed trials. The incidence of Lyme disease in the targeted communities ofSalisbury, Canaan, Cornwall, Westport and Weston were, respectively, 804.6,1202.6, 976.3, 221.4, and 129.5 per 100,000 in 2005. Tick densities in thesecommunities are high with lawn counts typically ranging up to 0.05 nymphs persquare meter for the entire tick season from May through July. Densities inwoodland areas are higher and peak densities in June easily exceed 0.1 m2.June peak densities at the residential sites are variable, but range fromapproximately 0.08 to 3.1 per m2. Additional properties, if needed, can be locatedwith the cooperation of the local health districts, with which we have a long, closeworking relationship. Several years of tick collection data will permit stratificationon randomization to minimize variability in tick abundance between treated anduntreated properties. We will measure the impact of the natural products on questing nymphs bydrag sampling for ticks and evaluating relative tick abundance in comparison tocontrols. The natural products will be applied to field and control plots treatedwith the same formulation with or without the active ingredient (AI) in early June.Drag samples will be performed before the acaricide application and weeklythereafter for at least one month and biweekly to the end of the nymphal tickseason (end of July). A border will be established around core areas treated withthe natural product to assess spatial repellency and both the core and border willbe sampled. The CDC project scientist will participate in the design of laboratoryand field studies to test natural acaricide activity. The test material will be applied by hydraulic sprayer to the sites by a licensedcommercial applicator using a high capacity hydraulic sprayer under thesupervision of the PI. Tick sampling will begin in late May to permit a treatmentpresample. Sites will be selected with regard to similar lot size and landscaping,and without recent or concurrent use of chemical acaricides. A single applicationrate (2% nootkatone in the first summer) will be used for the residential fieldtrials. Sites treated with the formulation without the natural product AI will serveas the positive control. Each of the 35-40 woodland edge plots will be assignedrandomly to one of four groups: 1. Due to high cost of nootkatone (2334.00/Kg of50% nootkatone) we plan ten homes (plots) will receive 2% nootkatone, 2. tenplots will receive the nootkatone formulation without the AI, serving as a positivecontrol, 3. five plots will receive a tank mixture of 0.1% nootkatone (exact ratesubject to further testing) and M. anisopliae (Tick-Ex)(see Objective 4), and 4. afinal set of ten plots will serve as negative controls, with no nootkatone or fungusapplied. Tick control and persistence of the nootkatone will be evaluated by flaggingthe plots treated with nootkatone, nootkatone and M. anisopliae, and the controlplots as mentioned previously. A subsample of ticks from the treated and controlplots will be held individually in vials at 25 C and > 90% RH for 4 weeks andchecked weekly for mortality. Additional dragging off-plot at the same residenceswill provide additional ticks for testing for B. burgdorferi. Infection of ticks with B.burgdorferi also will be determined by indirect fluorescent antibody (IFA) stainingand PCR techniques. Similar procedures will be used for subsequent year fieldtrials of encapsulated nootkatone formulations. The analysis of the tick data will be based on calculation of percent controland comparison of tick densities per 100 m2 between the treatments and control.The percent control of nymphs will be calculated on comparison of nymphabundance per 100 m2 area in control versus treatment sites. Percent (%) controlwill be calculated using a modification of Abbott's formula (Neal, J. W., 1976. Amanual for determining the small dosage calculations of pesticides andconversion tables. Entomological Society of America, College Park, Md.): % Control = 100 (Yc-Yt)/Ycwhere, Yc and Yt are the mean number of nymphs collected from control andtreated sites per 100 m2, respectively. The statistical analysis will be performedusing One Way ANOVA, Kruskal-Wallis One way of Variance on Ranks and TwoWay ANOVA (SigmaStat3.1, Systat Software Inc.) for comparing the number ofnymphs/100m2 collected from different treatment sites. The CDC project scientistwill participate in the evaluation of the data collected on tick control achieved byapplication of natural products. Residual/Degradation Studies Nootkatone - Because efficacy of thenootkatone and other natural products will depend, in part, on the residual effectsof the formulation, we will 1) determine how concentration changes over time andcompare residual activity of emulsifiable and extended duration formulationsunder semi-field conditions prior to full field studies and 2) conduct chemicalanalyses of the droplets placed outdoors in the study site during spray trials.Evaluation of the residues and acaricidal activity of extended release nootkatoneformulation under greenhouse conditions will provide information on theformulation, optimizing the formulation, and determining concentrationsnecessary for the full field trials. By collecting field samples that have beenoutdoors for varying periods of time, extracting and analyzing the compounds, weexpect to quantify the half-life of each formulation i.e., the time it takes to bereduced to one-half its original amount, and thereby determine how quickly itloses effectiveness when exposed to the environment. An extended durationformulation with good AI availability may provide longer control and allow use ofa lower and more economical concentration of nootkatone in the field withcomparable efficacy. Sample cards will be set in the leaf litter, flagged, and recovered at setintervals to evaluate by gas chromatograph/mass spectrometry (GC/MS)techniques the initial application of the nootkatone and residual activity over time.Samples of soil and leaf litter will also be collected for residual analysis.Preliminary analysis of a technical sample of nootkatone in the Department ofAnalytical Chemistry shows that GC/MS techniques are appropriate and suitablefor measuring nootkatone. The analysis using 98.0% nootkatone standard fromBedoukian Research shows that we can detect at least 0.5 ug/g of nootkatone(5x10-6 percent) prepared in solvent. However, extraction procedures for thenootkatone from the card and vegetative samples will need to be established.Samples will be analyzed using by gas chromatography (GC) using an Agilent6890 and a 5975 mass selective detector (MSD) operated in scan mode. The GCis equipped with a J&W Scientific DB-5MS+DG capillary column and an Agilentprogrammable temperature vaporization (PTV) inlet. This inlet is capable ofsolvent vent, split, and splitless injection techniques. The instrument can beoperated in selected ion monitoring mode to increase sensitivity if that isrequired. The presence of nootkatone will be confirmed by comparison to anauthentic neat stock standard purchased from Bedoukian Research. We also propose to examine product residual activity and degradation undersemi-field conditions in the greenhouse. Potted grass will be treated withextended release and emulsified formulations of nootkatone and placed ingreenhouse where temperature, humidity and solar radiation will be monitored.Grass samples will be collected at fixed intervals of time for chemical analysisand a tick Petri dish bioassay. The cut grass will be placed in petri plates, ticksintroduced for 24-h, and morbidity and mortality assessed. Nootkatone levels willbe analyzed by the Department of Analytical Chemistry by gas chromatography(GC) mass selective detector (MSD) techniques (i.e., GS/MS) (see detailedmethods in Objective 2).Objective 3. Test repellency of the natural products against I. scapularis in thelaboratory and in the field with treated and untreated flannel tick drags. Nootkatone and carvacrol, another component of the Alaska yellow cedarextract, has been shown to have significant repellent activity against I. scapularis,but carvacrol has not been tested in the field. Other possible natural compoundsfor repellent testing include nepetalactone (from catmint, Nepeta cataria), lemon-scented eucalyptus, Corymbia citriodora, oil, garlic-based product, and curcuminwith mustard oil (from turmeric, Curcuma longa, occasionally used as a tickrepellent in India). Catmint has demonstrated repellency against termites,mosquitoes, and cockroaches (Peterson et al. 2002; Peterson and Ems-Wilson2003; Bernier et al. 2005; Chauhan et al. 2005). A product containing 30%lemon-scented eucalyptus, Corymbia citriodora, oil and the oil itself was 74 and85% repellent, respectively, to I. ricinus in a similar blanket-dragging technique(Jaenson et al. 2006). Laboratory Tests (vertical tube tick assay) - Nootkatone, valencene-13-ol,and carvacrol have been shown to have good repellency against I. scapularis inthe laboratory and are already candidates for further evaluation in the field(Diethrich et al. 2006). We propose to also screen four materials; Repel(R) Lemon-Eucalyptus spray (Spectrum Brands, St. Louis, MO) containing lemon eucalyptusoil (p-methane-3,8-diol), garlic-based products, turmeric and mustard oil, andcatmint oil (nepetalactone), against I. scapularis. For these de novo compoundsfor which no peer-reviewed published data is available, initial evaluations will beconducted in the laboratory using a modified in vitro vertical tick repellencybioassay (Lerdhusnee et al. 2003; Carroll et al. 2004; Diethrich et al. 2006). Inbrief, test compound is pipetted onto a cotton-tipped applicator and mounted tothe bottom center of a 1-dram friction cap vial in non-toxic modeling clay. Thetreated area above a line is considered as the repellent zone. Single ticks areintroduced at set intervals after treatment. A screening cloth is placed over thevial to prevent the tick from escaping; a human hand is placed over the vial forstimuli. If a tick crosses above the line and remains in the repellent zone for > 5s,the tick is deemed nonrepelled by the test compound. Efficacy of individualcompounds will be determined by calculating a repellent dose - concentration50% value by probit analysis. The CDC project scientist will participate in theevaluation of the data collected on repellency against ticks achieved byapplication of natural products. Field Tests (drag cloth trials) - Laboratory tests do not fully measure thetick host-seeking behavior and host response. A tick repellent must operate attwo levels; prevent crawling across a treated surface (skin or clothing) and/orprevent attachment. In the field, flannel or fleece material will be treated with atleast two concentrations of nootkatone or carvacrol and dragged over thevegetation in areas infested with nymphal I. scapularis. If fewer ticks arecollected on the treated cloth, daily sampling will be done until repellency is nolonger observed. Field testing of other natural products (e.g., nepetalactone,valencence, lemon eucalyptus, or other compound as mutually identified by thePI and CDC) will be based upon repellency in laboratory screening.Objective 4. Test formulations of natural products, particularly nootkatone, in thelaboratory and field in combination with the entomopathogenic fungus, M.anisopliae Strain 52, for the control of I. scapularis nymphs. We propose to conduct both laboratory and field evaluations of the oil-basedM. anisopliae formulation (Tick-Ex') as a complementary or potentiallysynergistic agent with the natural product, nootkatone and other extracts fromAlaska yellow cedar, particularly carvacrol. The studies will address compatibilityof natural product and fungus and efficacy of lower concentrations of the naturalproducts when combined with the fungus. Laboratory Tests (Germination assay) - The natural products will initiallybe evaluated in the laboratory for compatibility with M. anisopliae at variousconcentrations (0.1-1%) suitable for field application. While inhibition of thefungus is observed with a nootkatone concentration of 1%, germination does notappear to be affected at lower concentrations. In the 'crawl' method we will use,nootkatone, for example, with concentrations 0.05, 0.1 and 0.5% combined withM. anisopliae (Tick-Ex; 3.9 x109 cfu/ml) at a rate of 3.2x105 cfu/cm2 in a 65 mmdia. Petri dish assay using Whatman No. 1 filter paper arena in 5 replications percombination with 10 I. scapularis nymphs per plate. Ticks will be exposed forspecific duration of time (e.g., 3 and 30 minutes and 1 hour). We will alsoexamine how encapsulating nootkatone may provide a barrier that will allowmixing lower concentrations of nootkatone with spores for application withoutinhibiting spore germination. Every week observations for mortality will berecorded. At the end of experiment, percent mortality will be calculated for ticksexposed to different concentrations for different intervals of time and numberscompared using the Kruskal-Wallis analysis of variance on ranks. Field Tests - In the field, at least 5 field plots will be treated with acombination of nootkatone and the fungus. Controls will be the same as thenootkatone without the AI. Collected ticks will be evaluated for development ofmycoses with M. anisopliae by holding for observation of fungal development.Entomopathogenic fungi will be provided by Novozymes Biologicals, Inc., Salem,VA. A combination of 0.1% nootkatone and Tick-Ex at a rate of 2.6 fl oz /1000 ft2(i.e., 3.2x105 cfu/cm2) will be applied as directed by the principal investigatorsusing a high volume hydraulic sprayer at 150 psi. Ticks will be collected asoutlined for the nootkatone. Ticks from the fungus treated sites and control plotswill be held in vials at 25 C and > 90% RH for 4 weeks and checked weekly formortality due to fungal infection.TIMELINEYEAR 1 (April 2008 - March 2009)Laboratory Tests (CAES)' Conduct germination assays for nootkatone and carvacrol for compatibility with M. anisopliae via Petri plate assays. (Compatibility of nootkatone and carvacrol/fungus testing is anticipated to be completed prior to start of project).' Conduct efficacy testing of new nootkatone formulations against I. scapularis (disposable pipette method) and nootkatone/fungus (Petri plate tick bioassays).' Conduct repellency testing of other natural compounds against I. scapularis (vertical tube tick repellent bioassay).Field Trials (CAES)' Conduct spray trials at residential sites with current emulsifiable concentrate (EC) 2% nootkatone formulation and with 0.1% nootkatone in combination with Metarhizium. Chemical analysis of samples for nootkatone residues.' Conduct repellency testing of nootkatone and carvacrol via drag cloth trials.Formulation Development (USDA-ARS)' Improve the current EC formulation. Evaluate the current EC for emulsification characteristics and determine if improvements are necessary to establish a standard EC formulation to compare with subsequent formulation samples.' Develop preliminary encapsulation techniques for nootkatone using A) coacervation, B) Fantesk encapsulation, and C) spray dried encapsulation' Provide samples for initial laboratory screening of formulation for efficacy against ticks.' Select representative samples for subsequent optimization based on efficacy, cost, and suitability for aqueous spray applications.YEAR 2 (April 2009 - March 2010)Laboratory Tests (CAES)' Conduct efficacy testing of encapsulated nootkatone formulation(s) against I. scapularis (disposable pipette method) and encapsulated/fungus (Petri plate tick bioassays).' Conduct germination assays for selected encapsulated nootkatone formulation(s) and new emulsifiable formulation(s) for compatibility with fungus (Petri plate assays).Field Trials (CAES)' Conduct field spray trial of a selected nootkatone encapsulated formulation(s) at residential sites.' Conduct residual/degradation study of any new EC and selected nootkatone encapsulated formulation(s) in the greenhouse with tick bioassay and nootkatone residue analysis.' Conduct repellency testing of eucalyptus commercial product and another compound based on year 1 results and discussion with CDC (drag cloth trials).Formulation Development (USDA-ARS)' Optimize selected encapsulation procedures based on encapsulation efficiency, maximized active load and mixing characteristics.' Provide formulation samples to evaluate optimized formulations relative to the previously established standard EC formulation.' Produce sufficient product for up to three samples for initial field evaluation' Establish formulation samples to evaluate storage stability.YEAR 3 (April 2010 - March 2011)Laboratory Tests (CAES)' Conduct efficacy testing of stored encapsulated nootkatone formulation(s) against I. scapularis (disposable pipette method) and encapsulated nootkatone/fungus (Petri plate tick bioassays).' Conduct germination assays for additional selected encapsulated nootkatone formulation(s) and new emulsifiable formulation(s) for compatibility with fungus (Petri plate assays).Field Trials (CAES)' Conduct field spray trial of another selected nootkatone encapsulated formulation at residential sites.' Conduct residual/degradation study of additional selected nootkatone encapsulated formulation(s) in the greenhouse with tick bioassay and nootkatone residue analysis (if needed).' Summarize data, and submit a final report, submit one or more manuscripts for publication of the laboratory tests and field trials.Formulation Development (USDA-ARS)' Evaluate storage stability of formulations for physical characteristics, mixing characteristics, and loss of active agent at quarterly time intervals for up to one year, and provide stored samples for efficacy evaluation.' Produce selected formulation product for field trials.' Summarize data, and submit a final report, submit one or more manuscripts for publication.PLAN FOR SHARING DATA The Connecticut Agricultural Experiment Station dissemination of any and alldata collected under the CDC data sharing agreement will be as follows: in atimely manner, completely, and as accurately as possible, to facilitate thebroader community, and developed in accordance with CDC policy on Releasingand Sharing Data as required by the program manager. The type of data collected in this study will be specific results of thelaboratory and field experiments and composition of any formulation devised forthe natural products. Data will be kept in MS Excel, analyzed with commercialstatistical programs, and summarized in MS Word documents for reportingpurposes. The data and results will be provided to CDC in required annualscheduled reports and at any time with the participating CDC Project Scientist.Data will be shared between project cooperators via e-mail, disk or any otherconvenient method. Letters on research results and progress will be sent to allparticipating landowners on the project. As a state government researchinstitution, all data and files at The Connecticut Agricultural Experiment Station,unless specifically exempted by state or federal law, are open to public access. CAES will distribute objective, unbiased, results of the trials to homeowners,the commercial applicator industry, and scientific community through variouswritten publications, including papers in peer-reviewed journals, presentations atindustry meetings and workshops, and other public forums. Both nootkatone (andother botanical products) and M. anisopliae would meet current organic land usestandards. Information on these products would be especially well received bythe organic land care industry. CAES has provided tick control information (i.e.landscape modifications, deer exclusion, least-toxic tick control, etc.) to membersof the Northeast Organic Farming Association (NOFA) at special workshops forseveral years. The PI's Tick Management Handbook has been used bythousands of stakeholders. The 10,000 copies printed in 2004 have been widelydistributed and in 2006, there were 117,000 downloads of all or part of thehandbook from the CAES website. A printing of a new edition is currently inprogress. Results of the studies will be disseminated through public talks,presentations at scientific meetings, and submission to peer-reveiwed journals.IMPACT New IPM strategies incorporating the application of natural botanicalcompounds such as nootkatone and entomopathogenic fungi would receivebroad support and use from those practicing organic land care, in schoolenvironments that mandate IPM, and other high risk areas where chemicals areless acceptable, thereby reducing the risk of tick-associated disease andpesticide use. The commercial availability of natural products and biopesticideswould provide a natural alternative to synthetic chemical acaricides. In addition,improved tick management could be obtained at sites that currently do not allowor restrict chemical pesticides. Although chemical pesticides are banned atschools in Connecticut except for public health risks (i.e., mosquitoes, ticks,fleas), restrictions on use continue to increase. Availability of a nootkatone or other natural product-based tick controlformulation and M. anisopliae should result in decreased pesticide use andincreased implementation of IPM for tick control. CDC funded surveys haveshown that landscape modifications are the most popular option in tick IPM, only22-38% of residents spray chemical insecticides for tick control, 69% of residentsoriginally indicated that chemicals were not an option despite their effectivenessa.Chemicals applications for tick control comprise a relatively small part ofresidential agrichemical use, but tick control business for land care businesses isgrowing (Stafford 1997). A 2005 follow-up survey of commercial applicators inConnecticut found that 57% offering tick control would use a fungus biopesticideif available and effectiveb. A third of commercial applicators indicated they getrequests for alternatives to insecticides. Presumably at least a similar proportionwould offer a natural chemical to their clients. The development of naturalproducts and the availability of a commercial biocontrol agent can be importantcomponents of an integrated tick management program. These products couldoffer a less toxic natural and biological alternative, potentially workingsynergistically, to synthetic chemicals for the control of nymphal I. scapularis.aKnowledge, Attitudes and Behavior (KAB) Survey in the Westport WestonHealth District, CT Department of Public Health, as part of a CDC-fundedcommunity Lyme disease prevention projects, 2000-2007.bStafford, unpublished data from 2005 statewide follow-up survey of licensedcommercial pesticide applicators.EVALUATION Project progress will be evaluated through an internal review process withconsultations between the investigators and reports to CDC. A HHS/CDC projectscientist will have substantial programmatic involvement in the proposed studies.In addition, consultations will be conducted with the company with the fungal tickcontrol product. Written progress reports to the CDC will be submitted on therequired schedule. Therefore, meetings between the principal investigators,collaborators, and other parties, submission of progress reports, and consultationwith the CDC will provide ongoing evaluation of the project. If progress is lessthan anticipated, a discussion will ensue concerning the reasons why socorrections made. Changes to the work plan will be discussed with the CDC.Progress on the project will be based on the successful completion of theobjectives, impact on I. scapularis, and ultimately on the introduction of productas a tool within an integrated tick management framework.G. HUMAN SUBJECTS RESEARCH No human subjects are proposed in this research.F. VERTIBRATE ANIMALS The CAES adheres to the Animal Welfare Act, the PHS Policy on the Careand Use of Animals, and the Guide to the Care and Use of Laboratory Animals.This institution is licensed by the USDA (Registration No. 16-R-0039, expirationdate 07/07/09) and has an animal welfare assurance statement on file with theOffice for Protection from Research Risks, National Institutes of Health (#A4050-01). The institutional animal care and use committee must approve all work withanimals. An IACUC approved protocol for the rearing of Ixodes scapularis tickson animals is on file at CAES. The animals to be used in the proposed study are for maintaining a tickcolony of I. scapularis. The ticks will be used for efficacy testing of the naturaland entomopathogenic fungal product. There are no viable in vitro methods yetavailable for successfully rearing ticks. Immature stages (larvae or nymphs) of I.scapularis are fed on the selected strain of laboratory mouse (C3H or Swissmice). Adult ticks are fed on the ears of a New Zealand White rabbit. While thenumber of animals used will depend upon the total number of adult ticks to be fedto supply larval ticks from ovipositing females, we anticipate using 20-40 miceand 2-4 rabbits as approved in the protocol. Tick feeding does not involve morethan minimal or momentary pain or distress to animals or restraint (USDACategory C), and therefore, no anesthetic, analgesic, or tranquilizer is required.Veterinary care is provided by the attending veterinarian, Kimberly McClureDVM. Euthanasia of mice is by carbon dioxide via anesthesia chamber oroverdose of inhalation anesthesia (isoflurane, methoxyflurane) and, for rabbits,by barbiturates administered by a veterinarian consistent with recommendationsof the American Veterinary Medical Association.
