Laboratory and field evaluation of five repellents against the black flies Prosimulium mixtum and P. fuscum (Diptera: Simuliidae).

Deet (N,N-diethyl-3-methyl-benzamide), the lactone CIC-4 ([2-hydroxy-methyl-cyclohexyl] acetic acid lactone), the USDA Proprietary Chemicals AI3-37220 (1- [3-cyclohexen-1-ylcarbonyl]-2-methylpiperidine) and ++[3-35765 (1-[3-cyclohexen-1-ylcarbonyl] piperidine), and the U.S. military extended duration repellent formulation (EDRF) of deet were evaluated for repellency in the laboratory and field against the black flies Prosimulium mixtum and P. fuscum. CIC-4, AI3-37220, and AI3-35765 were as effective as deet at repelling P. mixtum and P. fuscum in laboratory and field experiments. Only the EDRF provided significantly longer protection than the deet standard against these black flies in the field.     

Comparative resistance of Anopheles albimanus and Aedes aegypti to N,N-diethyl-3-methylbenzamide (Deet) and 2-methylpiperidinyl-3-cyclohexen-1-carboxamide (AI3-37220) in laboratory human-volunteer repellent assays

The insect repellents N,N-diethyl-3-methylbenzamide (Deet) and the racemate and 1S,2'S stereoisomer of 2-methylpiperidinyl-3-cyclohexene-1-carboxamide (AI3-37220) were tested against Anopheles albimanus Wiedemann and Aedes aegypti (L.) in laboratory human-volunteer assays. Estimated skin doses of Deet or racemic AI3-37220 required to reduce biting by 95% in Ae. aegypti were 2.3 and 3.5 x 10(-2) micromol/cm2 skin, respectively, whereas estimated doses for 95% bite reduction of An. albimanus in an approximately 40-yr-old laboratory colony established from El Salvador were 5 times higher at 12 x 10(-2) micromol Deet/cm2 skin and >20 x 10(-2) micromol/cm2 skin for AI3-37220. In tests with the 1S,2'S stereoisomer of AI3-37220, a newly established colony of An. albimanus from Belize bit less aggressively than El Salvador An. albimanus. However, the Belize-derived mosquitoes were as resistant as the old El Salvador colony to repellent effects of 1S,2'S stereoisomer of 2-methylpiperidinyl-3-cyclohexene-1-carboxamide. Earlier workers surmised that usual skin doses of Deet would offer only limited protection against An. albimanus in the field. Our findings support this speculation, but they also indicate that doses of Deet higher than those needed for protection against Ae. aegypti might offer reasonable protection against An. albimanus. Results indicate that neither racemate nor 1S,2'S stereoisomer of 2-methylpiperidinyl-3-cyclohexene-1-carboxamide offer as much protection as Deet against An. Albimanus, despite being highly effective against Ae. aegypti.     

Field evaluation of deet and a piperidine repellent against Aedes communis (Diptera: Culicidae) and Simulium venustum (Diptera: Simuliidae) in the Adirondack Mountains of New York

Repellent efficacy of N,N-diethyl-3-methyl-benzamide (deet), the piperidine, 1-[3-cyclohexen-1-ylcarbonyl]-2-methylpiperidine (AI3-37220), and a 1:1 ratio of deet + AI3-37220 were evaluated topically (0.25 mg/cm2 applied in ethanol solution) on human volunteers against the mosquito Aedes communis (DeGeer) and the black fly Simulium venustum Say. The average repellency of all three formulations was > 95% at 4 h. For both mosquitoes and black flies, deet alone provided < 90% protection at 6 h, whereas AI3-37220 provided > 95% protection. Although repellent treatments were not significantly different overall, the contrasts between AI3-3720 versus deet were significant at 6 and 8 h. The 95% confidence interval on percent repellency at 6 h ranged from 90.1 to 98.9% for AI3-37220 versus 64.3 to 82.2% for deet, and at 8 h ranged 76.1 to 88.5% for AI3-37220 versus 47.8 to 64.0% for deet. Similarly, the confidence interval for protection against black flies at 6 h by (AI3-37220 ranged from 86.3 to 99.5% and did not overlap with the confidence interval provided by deet alone (51.2 to 78.8%). There was no evidence of synergistic repellency from a combination of the two compounds; i.e., protection from combined compounds was no better than either repellent used alone.     

Efficacy of plant-derived and synthetic compounds on clothing as repellents against Ixodes scapularis and Amblyomma americanum (Acari: Ixodidae)

We conducted field trials to compare the relative repellent activity of two natural product compounds (nootkatone and carvacrol) with commercially available plant-derived (EcoSMART organic insect repellent) and permethrin-based (Repel Permanone) repellents against adult Ixodes scapularis Say and Amblyomma americanum (L.) (Acari: Ixodidae) by using treated coveralls. One day after treatment, nootkatone and carvacrol provided 100% repellency of I. scapularis adults, with nootkatone maintaining complete protection through 3 d, whereas carvacrol showed steadily declining repellency against I. scapularis during the 7-d course of the trials. Nootkatone was at least as effective against host-seeking A. americanum as against I. scapularis through 3 d. Carvacrol provided little protection against A. americanum adults. Both natural compounds performed well initially in comparison with the commercial products. After 7 d, nootkatone was the most effective against both species followed in order of activity by Permanone, EcoSMART, and carvacrol. Nootkatone seems to have offer considerable potential as a clothing repellent against both I. scapularis and A. americanum.     

Relative abundance and prevalence of selected Borrelia infections in Ixodes scapularis and Amblyomma americanum (Acari: Ixodidae) from publicly owned lands in Monmouth County, New Jersey

To evaluate their potential importance in the transmission of ixodid tick-borne borrelioses in Monmouth County, NJ, we collected host-seeking Ixodes scapularis Say and Amblyomma americanum (L.) (Acari: Ixodidae) adults and nymphs to determine relative encounter frequencies and the infection prevalence of selected Borrelia spp. in their respective tick vectors. We also reviewed records of all ticks submitted for identification by the public in Monmouth County during 2001-2005. Relative abundance of the two species varied markedly among sites. Adult encounter frequencies for the two species were similar; however, A. americanum nymphs were encountered 3 times more frequently than I. scapularis nymphs. Of 435 ticks submitted by the public, 50.1 and 38.9% were I. scapularis and A. americanum, respectively. However, during May through August, the peak Lyme disease transmission season in New Jersey, significantly more submitted ticks were A. americanum (55.9%), compared with I. scapularis (34.1%). Polymerase chain reaction analysis of 94 1. scapularis and 103 A. americanum adults yielded infection prevalences of 31.9% for B. burgdorferi and 5.8% for B. lonestari, respectively. Although the infection prevalence of B. burgdorferi in I. scapularis was considerably higher than the infection prevalence of B. lonestari in A. americanum, the higher encounter frequencies for A. americanum compared with I. scapularis observed in this and other studies may result in increased risk of acquiring exposure to A. americanum-transmitted pathogens. The potential public health implications of these results are discussed.     

Repellent activity of fractioned compounds from Chamaecyparis nootkatensis essential oil against nymphal Ixodes scapularis (Acari: Ixodidae)

Preliminary repellent activity of 14 natural products isolated from essential oil components extracted from the heartwood of Alaska yellow cedar, Chamaecyparis nootkatensis (D. Don) Spach., were evaluated against nymphal Ixodes scapularis Say in a laboratory bioassay and compared with technical grade N,N-diethyl-3-methylbenzamide (deet). Four hours after treatment, nootkatone and valencene-13-ol had repellent concentration (RC)50 values of 0.0458 and 0.0712% (wt:vol), respectively; two additional Alaska yellow cedar compounds, nootkatone 1 --> 10 epoxide and carvacrol had reported RC50 values of 0.0858 and 0.112%, respectively. The observed RC50 value for deet was 0.0728% (wt:vol). Although not statistically significantly more active than deet, the ability of these natural products to repel ticks at relatively low concentrations may represent a potential alternative to synthetic commercial repellents.     

Different activities and footwear influence exposure to host-seeking nymphs of Ixodes scapularis and Amblyomma americanum (Acari: Ixodidae)

The relative potential for a person accidentally acquiring host-seeking nymphs of the blacklegged tick, Ixodes scapularis Say, and lone star tick, Amblyomma americanum (L.), while wearing either of two types of footwear, walking, crawling on hands and knees, and sitting on large fallen logs in deciduous woods, was evaluated. Although flag samples indicated substantial populations of I. scapularis nymphs and low to moderate numbers of A. americanum at the study sites, relatively few I. scapularis and fewer still A. americanum nymphs were acquired during 30-s and 5-min walks. Significantly fewer I. scapularis were picked up when boots were, worn with ankles taped (an anti-tick precaution) than when sneakers were worn with socks exposed during 5-min walks, but when thus attired, there was no significant difference between the number of nymphs acquired during 30-s walks. Nymphs of I. scapularis did not appear to accumulate incrementally on footwear or clothing during walks when boots were worn and ankles taped. Crawling for 30 s (approximately 3 m distance) yielded significantly more I. scapularis nymphs than walking for 30 s. During crawling, I. scapularis nymphs were picked up on 58% of the 30-s samples. Most ticks picked up during crawls were on pant legs. When a flannel flag cloth (0.5 by 0.5 m) was appressed to the upper surface of logs suitable to be sat upon by tired hikers, I. scapularis nymphs were found on 87% of the logs and in 36% of the samples. These data indicate that the potential for contact with host-seeking nymphs of I. scapularis occurring at these densities is greatly elevated by engaging in activities that involve contact with fallen logs and close contact of hands and knees with leaf litter.     

Novel arthropod repellent, BioUD, is an efficacious alternative to deet

For >50 yr, N,N-diethyl-3-methylbenzamide (deet) has been the standard for arthropod repellents and has been an important tool to protect people from disease agents carried by ticks, mosquitoes, and other arthropods. However, some people avoid using deet because of concerns about adverse health effects. In 2007, a new repellent, BioUD, with the active ingredient 7.75% 2-undecanone, originally derived from wild tomato (Lycopersicon hirsutum Dunal f. glabratum C. H. Müll) plants, was registered by the U.S. Environmental Protection Agency. In the current study, repellent efficacy of BioUD was compared using arm-in-cage studies with 7 and 15% deet against the mosquitoes Aedes aegypti (L.) and Aedes albopictus Skuse. No differences were found in mean repellency over 6 h after application between BioUD versus 7 and 15% deet for Ae albopictus. For Ae. aegypti, no differences were found over the same time period for 7% deet. Compared with 15% deet, BioUD mean repellency was lower over the 6-h test period. Human subject field trials were conducted in North Carolina, United States, and Ontario, Canada, comparing the repellency of BioUD to products containing 25 and 30% deet. BioUD provided the same repellency or was more efficacious than 25 and 30% deet, respectively, in these studies. Laboratory trials were conducted to determine the repellent activity of BioUD against the American dog tick, Dermacentor variabilis (Say), on human skin and cloth. BioUD repelled ticks at least 2.5 h after application to human skin. On cloth, no differences in mean repellency were found through 8 d after application between BioUD and 7% deet. In a two-choice test for BioUD versus 15% deet on filter paper, ticks spent significantly more time on the deet-treated surface than the BioUD-treated surface. Based on these studies in toto, BioUD is an efficacious alternative to deet in its repellent activity.     

A new module for quantitative evaluation of repellent efficacy using human subjects

A new module for quantitative evaluation of arthropod repellents in human subjects was designed, constructed, and protocols for use of the module were developed. Doses of 3 arthropod repellents, 1-[3-cyclohexen-1-ylcarbonyl] piperidine (AI3-35765), 1-[3-cyclohexen-1-ylcarbonyl]-2-methylpiperidine (AI3-37220), and N,N-diethyl-3-methylbenzamide (deet), were evaluated using the mosquito, Anopheles stephensi Liston. Biting responses to varied doses of the repellents on human skin were quantified, and the effectiveness of the 3 repellents was compared. The new module consists of 6 test cells and permits the simultaneous comparison of up to 5 repellent doses or chemical types and a control using a complete randomized block design with minimal treatment interaction and with > or = 6 replicates per human subject.     

Ticks parasitizing dogs in northwestern Georgia

From January 1998 through September 1999, 324 dogs in three northwestern Georgia counties were examined for ticks. Six species of ticks were recovered. The three most commonly collected ticks were the American dog tick, Dermacentor variabilis (Say) (310 male male, 352 female female; prevalence, 97%; mean intensity 2.1); the brown dog tick, Rhipicephalus sanguineus (Latreille) (118 male male, 119 female female, 38 nymphs; prevalence, 22%; mean intensity, 3.8); and the lone star tick, Amblyomma americanum (L.) (8 male male 26 female female, 2 nymphs; prevalence, 5%; mean intensity, 2.4). Other ticks recovered were Ixodes cookei Packard (3 female female); the Gulf Coast tick, Amblyomma maculatum Koch (2 female female); and the blacklegged tick, Ixodes scapularis Say (1 female). Another adult female specimen of I scapularis was recovered from a cat, further reinforcing that this medically important tick is present in northwestern Georgia.     

Seasonal distribution and abundance of ticks (Acari: ixodidae) in northwestern Florida

A 2-yr study was conducted in a northwestern Florida state park and recreation area to determine tick species composition, seasonal abundance, and spatial distribution. Risk of tick attachment to park visitors was also assessed relative to tick abundance from several habitats. Tick collections consisted of weekly cloth drag samples obtained from ground and emergent vegetation along a deer trail, a walking trail located in a 3-mo-old control burned area of forest, a nonburned walking trail, picnic area, and campsites. Sampling was conducted from February 1994 through February 1996. Ticks collected during the study were as follows in descending order of abundance: Ixodes scapularis Say, Amblyomma americanum (L.), A. maculatum Koch, and Dermacentor variabilis (Say). Significantly more (<0.05) adult ticks were recovered from vegetation compared with ground samples only for I. scapularis. Adult questing I. scapularis were collected from October through May (peak December). A. americanum adults were collected from March through August (peak May). Adult A. maculatum were collected during August and September and adult D. variabilis were collected July and August. Larval and nymphal stages of questing A. americanum were collected from June through November (peak July) and February through October (peak September), respectively. A. maculatum nymphs were collected from February through April, June, and September (peak March). No nymphs of I. scapularis were collected. Only four larvae of D. variabilis were collected during the study, all during February 1995. No I. scapularis or A. maculatum larvae were collected from vegetation or ground drags. The greatest risk for tick attachment was in campsites where approximately 60% of all adult and nymphal host-seeking ticks were collected. The next greatest risk area was the walking trail located in the control burned area where an additional 28% of ticks were obtained.     

A cautionary note: survival of nymphs of two species of ticks (Acari: Ixodidae) among clothes laundered in an automatic washer

Host-seeking ticks often remain on clothing of persons returning home from work or recreation in tick habitats, and can pose at least a temporary risk to people and pets in these homes. Laundering clothing has been one of the recommendations to reduce tick exposure. Host-seeking lone star tick, Amblyomma americanum (L.), and blacklegged tick, Ixodes scapularis Say, nymphs confined in polyester mesh packets, were included with laundry in cold, warm, and hot wash cycles of an automatic clothes washer. Ticks were also placed with washed clothing and subjected to drying in an automatic clothes dryer set on high heat and on air only (unheated). Most nymphs (> or = 90%) of both species survived the cold and warm washes, and 95% of A. americanum nymphs survived the hot wash. At the time of their removal from the washer, I. scapularis nymphs were clearly affected by the hot wash, but 65% were considered alive 20-24 h later. Large percentages of nymphs of both species survived hot washes in which two other detergents (a powder containing a nonchlorine bleach and a liquid) were used. All ticks were killed by the 1 h cycle at high heat in the clothes dryer, but with unheated air some nymphs of both species survived the 1 h cycle in the dryer. Given the laundering recommendations of clothing manufacturers and variation in the use automatic clothes washers, laundry washed in automatic washers should not be considered free of living ticks.     

Repellent efficacy of DEET and KBR 3023 against Amblyomma hebraeum (Acari: Ixodidae)

The bont tick, Amblyomma hebraeum Koch, is the principal vector of Rickettsia africae, the agent of African tick bite fever, in southern Africa. In contrast to most other hard ticks, members of the genus Amblyomma exhibit a characteristic attack host-seeking behavior. The efficacy of topically applied 20% lotions of DEET (diethyl-3-methylbenzamide), the currently considered reference repellent substance, and KBR 3023, a recently developed piperidine compound, was evaluated against laboratory-reared A. hebraeum nymphs on treated fingers. Both substances repelled >85% of nymph attacks at 0 and 1 h postapplication. At 2, 3, and 4 h, the repellent efficacies of DEET were 84%, 68% and 71%, whereas those of KBR 3023 were 56, 55, and 54%. The observed differences between the two test substances were statistically significant only at 2 h postapplication. This first study of topical repellents against A. hebraeum suggests that 20% lotions of DEET and KBR 3023 are effective for 2 and 1 h, respectively, but that the repellent efficacies decrease thereafter.     

A survey for nymphs of host-seeking Ixodes scapularis and Amblyomma americanum (Acari: Ixodidae) on tree trunks in deciduous forests

Trunks of 83 trees in a mixed deciduous forests in Maryland were sampled for the presence of nymphs of the blacklegged tick, Ixodes scapularis Say, and the lone star tick, Amblyomma americanum (L.). Although one or more nymphs of either I. scapularis or A. americanum was found in leaf litter and substrate < or = 1 m from the bases of 47% of the trees sampled, a total of 6 I. scapularis nymphs was found on the trunks of only five trees. No nymphs were found on the trunks of 12 dead trees. No A. americanium nymphs were found on any tree trunks. The trunks were sampled to 2.5 m above the soil, but the nymphs were found < or = 1 m from the ground. More than 50% of I. scapularis nymphs found in the leaf litter < or = 1 m from bases of living trees were north of the trees sampled, whereas few I. scapularis were found west of trees. These findings suggest that the I. scapularis nymphs' presence on tree trunks is of little ecological consequence, unless nymphs were being removed from tree trunks by acquiring hosts at such a rapid rate that nymphal numbers on trunks could not accrue.     

Meteorologically mediated diurnal questing of Ixodes scapularis and Amblyomma americanum (Acari: Ixodidae) nymphs

Questing Ixodes scapularis Say and Amblyomma americanum (L.) nymphs were collected in the field by drag sampling to determine whether ticks would be collected in greater numbers during certain times of the day and under certain ambient meteorological conditions. Ticks were collected hourly for 16 h on 4 d and counts were contrasted with simultaneous measurements of ambient temperature and relative humidity, and with similar measurements made within the leaf litter. Peak numbers of questing I. scapularis and A. americanum nymphs were collected at distinctly different times of day, suggesting that the two species were responding to different environmental conditions. Both species demonstrated responses to diel changes in local meteorological conditions. Numbers of ticks were correlated with ambient temperature and humidity and with conditions recorded in the leaf litter that appeared to play a significant role in mediating questing behavior of both species. The time of day when tick sampling is conducted might significantly bias population estimates for sympatric species and the assessment of tick-borne disease transmission risk.     

Responses of three species of adult ticks (Acari: Ixodidae) to chemicals in the coats of principal and minor hosts.

In laboratory bioassays, host-seeking adult blacklegged ticks, Ixodes scapularis Say, lone star ticks, Amblyomma americanum (L.), and American dog ticks, Dermacentor variabilis (Say), were exposed to substances from the pelage of dogs and white-tailed deer, Odocoileus virginianus (Zimmermann). Male A. americanum exhibited an arrestant response to all samples of deer tarsal gland substances tested, whereas female A. americanum, female I. scapularis, and male and female D. variabilis responded to samples from certain deer and not others. Female I. scapularis, D. variabilis, and A. americanum of both sexes also showed an arrestant response to substances rubbed from the dorsal surface of dogs' ears. These findings suggest that, although these 3 species of ticks are associated with certain host species as adults (A. americanum and I. scapularis with white-tailed deer, D. variabilis with dogs), their repertoires of host-finding behaviors are broad, and use of minor host species may not be purely accidental.     

Lack of spatial autocorrelation in fine-scale distributions of Ixodes scapularis (Acari: Ixodidae)

Spatial patterns of Ixodes scapularis Say, the vector of the Lyme disease agent, have been examined at various geographic scales, demonstrating that distributions of these ticks are spatially autocorrelated at both national and state scales. We tested the hypothesis that distributions of nymphal I scapularis ticks at the fine scale of an endemic community also are spatially autocorrelated. Nymphal tick densities were determined by collecting ticks from 51 and 47 wooded residential properties in a southern Rhode Island town in 2002 and 2003, respectively. The average tick density at residences during 2002 was 51.17 ( +/- 46.04) nymphs per hour, with a range of 3-297 and median of 40.82. In 2003, the average tick density was 44.48 (+/- 38.31) nymphs per hour, with a range of 3-153 and median of 36. Semivariance analysis revealed no spatial autocorrelation in tick densities between residences, likely due to the high variability of tick distributions at this scale. Further analysis of drag-sampling data at individual residences by using Lloyd's patchiness index (m*/m) demonstrated a patchy distribution of nymphs. High variability of nymphal I. scapularis densities may greatly affect predicting spatial patterns of ticks at a fine scale.     

Comparative field evaluation of permethrin and deet-treated military uniforms for personal protection against ticks (Acari)

The effectiveness of permethrin-impregnated (PI) versus permethrin-sprayed (PS) military battle dress uniforms was evaluated in protecting soldiers from tick bite. At the same time, an extended-duration formulation of N,N-diethyl-m-toluamide (deet), when applied to clothing only, was evaluated and compared with the permethrin-treated uniforms. Testing consisted of exposing subjects, clothed in either untreated or the variously treated uniforms, to field populations of ticks during a series of 30-min field trials over a period of 21 d during July and August 1988. Permethrin, applied as either a spray (0.5% [AI]), or as an impregnant (0.125% [AI]/cm2), was more effective than deet (33.25% [AI]) applied to clothing only in protecting individuals from tick bite. The mean numbers of ticks on deet-treated, PI, and PS uniforms were 60, 97, and 98% lower, respectively, than on untreated uniforms against all encountered life stages of Amblyomma americanum (L), Dermacentor variabilis (Say), and Ixodes dammini Spielman, Clifford, Piesman and Corwin.     

Attempted transmission of Borrelia burgdorferi (Spirochaetales: Spirochaetaceae) (JDI strain) by Ixodes scapularis (Acari: Ixodidae), Dermacentor variabilis, and Amblyomma americanum.

Laboratory-reared Ixodes scapularis Say, Amblyomma americanum (L.), and Dermacentor variabilis (Say) were fed on New Zealand white rabbits experimentally infected with Borrelia burgdorferi (JDI strain). At repletion, spirochetes could be detected by dark-field microscopy only in I. scapularis. Acquisition rates were 18 and 21%. When previously exposed nymphs of each species were fed on susceptible rabbits, I. scapularis was the only tick of the three species that transmitted B. burgdorferi. When a single rabbit was experimentally infected with B. burgdorferi and infested at 7-d intervals with I. scapularis, A. americanum, D. variabilis, and a second time with I. scapularis, B. burgdorferi was detected again only in cultures from the two groups of I. scapularis. When molted nymphs from each tick species were allowed to feed on susceptible rabbits, spirochetes again were isolated only at necropsy from the rabbits on which the two groups of I. scapularis fed.     

Pathogenicity of entomopathogenic fungi Beauveria bassiana and Metarhizium anisopliae to Ixodidae tick species Dermacentor variabilis, Rhipicephalus sanguineus, and Ixodes scapularis

Nymphal and adult ticks from three different tick species, Dermacentor variabilis Say, Ixodes scapularis Say, and Rhipicephalus sanguineus Latrielle, were treated with conidia and blastospores of the entomopathogenic fungi Beauveria bassiana (Bals.) Vuill. and Metarhizium anisopliae Metschnikoff. Dose-response experiments indicated that a critical concentration of fungal spores is required for infection and mortality. Over a 28-d time course, fungal suspensions of either B. bassiana or M. anisopliae at 10(8) conidia/ml resulted in 50-70% mortality in adult I. scapularis and R. sanguineus, but <20% mortality in D. variabilis ticks. R. sanguineus nymphs were highly susceptible to both entomopathogenic fungi, displaying >60% mortality within 14 d postinfection and >90% mortality within 21-28 d postinfection. D. variabilis nymphs also were more susceptible than their corresponding adults, displaying mortalities ranging from 20 to 40% 28 d postinfection. I. scapularis nymphs, however, seemed to be slightly less susceptible than adults (45% mortality, 28 d postinfection). The addition of nutrients to fungal cell suspensions did not have any noticeable effects on mortality toward any of the tick species tested. Significant mortality against D. variabilis adults (approximately 65%) was noted only when B. bassiana fungal cells with growth media carryover were used as the inoculum against the ticks. Entomopathogenic fungi such as B. bassiana and M. anisopliae may have the potential for controlling populations of I. scapularis and R. sanguineus, and under certain conditions D. variabilis. Our results indicate that inoculum conditions can greatly affect successful virulence and subsequent mortality.