INTRODUCTION

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In February 1999, the government of the province of British Columbia, Canada, authorized an aerial spray program to control insurgent populations of the European gypsy moth, Lymantria dispar, found in and around the cities of Victoria, Duncan, and Nanaimo, British Columbia, Canada. The provincial government felt that the presence of gypsy moth populations posed a serious economic threat to exports of British Columbian lumber products to other provinces of Canada, as well as to neighboring states of the United States. Both a potential embargo of British Columbian lumber products and a significant environmental threat would exist if the insurgent gypsy moth populations were to become established. The biological insecticide Foray 48B, which contains spores of Bacillus thuringiensis subsp. kurstaki, was applied to more than 13,400 ha on Vancouver Island, of which 12,204 ha were in the greater Victoria region (Fig. 1).

View larger version (39K): [in this window] [in a new window]   FIG. 1.   Map of the spray application zone and the locations at which samples were taken. The boundaries of the spray zone are indicated by the light-gray line. Air samples and nasal swabs taken inside the spray zone are indicated as darker-gray circles; air samples and nasal swabs samples taken outside the spray zone are indicated as darker-gray squares. North is at the top of the figure. Prevailing winds at the time of spray were to the northeast. A-1, A-2, B-1, and B-2 indicate subzones covered by the airplanes during the application of Foray 48B.

B. thuringiensis is a spore-forming, gram-positive bacterium that produces intracellular, crystal proteins during sporulation. These parasporal proteins have insecticidal activity that are specific to certain groups of insects (27). The genus Bacillus has been divided into five groups (4). Group I contains a large number of soil-dwelling species, including B. thuringiensis, Bacillus sphaericus, Bacillus subtilis, Bacillus anthracis, and Bacillus cereus. Some of these bacilli are very closely related and form separate groups within the group I Bacillus species. The B. cereus group consists of B. cereus, B. anthracis, Bacillus mycoides, and B. thuringiensis, with B. cereus, B. mycoides, and B. thuringiensis being so closely related that some systematists consider the three species to be subspecies of B. cereus sensu lato (21). Analysis of the organization of the genomes of various strains of B. cereus and B. thuringiensis by pulsed-field electrophoresis revealed that some strains of B. cereus are more similar to certain strains of B. thuringiensis than they are to other strains of B. cereus, suggesting that these two species may actually be variants of the same species (9). At this time, however, B. thuringiensis stands as a separate species with distinct biochemical and genetic characteristics (20).

Because of the similarities between B. thuringiensis and B. cereus, and because B. cereus has been associated with food-borne outbreaks of gastrointestinal disease (13), B. thuringiensis-based products have been subjected to extensive evaluation both prior to and subsequent to becoming commercially available (22). In their review of human and laboratory data, McClintock et al. (22) concluded that B. thuringiensis subspecies are neither toxic nor pathogenic to mammals, including humans. Animal experimentation, however, has shown that intraperitoneal injection of B. thuringiensis can cause death in guinea pigs (14) and that pulmonary infection can result in the deaths of immunocompromised mice (18).

These experiments, however, reflect neither the types nor the consequences of human exposure to B. thuringiensis subsp. kurstaki, and reports of human disease caused by B. thuringiensis subsp. kurstaki are uncommon. A corneal ulcer developed in a previously healthy 18-year-old farmer who accidentally splashed a commercial B. thuringiensis product into his eye (26). Multiple thigh and knee abscesses containing B. thuringiensis were found in a previously healthy soldier who was severely wounded by a landmine explosion (17).

Epidemiological studies have been conducted on several occasions after aerial applications of B. thuringiensis subsp. kurstaki over populated areas. The Oregon State Health Division conducted a health surveillance study to assess the impacts of a spray program conducted in Lane County, Oreg., in 1985 to 1986. Fifty-five of 95 Bacillus isolates were identified as B. thuringiensis subsp. kurstaki. The bacteria were identified by microscopic examination, a technique which is currently not considered to be conclusive as it can result in a high proportion of misidentified bacteria. Upon further examination, 52 of the 55 isolates were assessed to be probable contaminants. Of the three remaining isolates, B. thuringiensis subsp. kurstaki could neither be ruled in nor be ruled out as a cause of the patient's disease (16).

A health impact surveillance study conducted in Auckland, New Zealand, during a white-marked tussock moth control program recovered B. thuringiensis subsp. kurstaki from three clinical isolates (3). Clinicians concluded, however, that B. thuringiensis subsp. kurstaki was not causally associated with disease and that all three cases represented sample contamination. Health impact surveillance programs were conducted in British Columbia, Canada, and in the states of Washington and Oregon in response to a 1992 aerial spray campaign. B. thuringiensis subsp. kurstaki was isolated from none of the samples collected in Washington, from 1 sample collected in Oregon, and from 325 samples collected in the lower mainland of British Columbia (1, 2, 23; M. A. Noble, P. Kandola, M. Amos, P. Riben, G. Cook, and C. Shaw, Abstr. 94th Gen. Meet. Am. Soc. Microbiol., p. 427, abstr. Q-224, 1994). All B. thuringiensis subsp. kurstaki isolates were determined to be the result of sample contamination and not causal agents of infection (1, 23; Noble et al., Abstr. 94th Gen. Meet. Am. Soc. Microbiol. 1994).

Aerial applications of Foray 48B were carried out on 9 to 10 May, 19 to 21 May, and 8 to 9 June 1999 to control European gypsy moth populations in Victoria, British Columbia. A coordinated study of the short-term health effects of the application of Foray 48B on the human population in Victoria, British Columbia, was undertaken by the Capital Health Region during this period (8). The study included a survey of the health of asthmatic children in the region, a survey of the general health of the population, monitoring and analysis of visits to physician's offices and hospital emergency departments, a review of self-reported complaints of health symptoms made to telephone information and support lines, clinical surveillance of patients with infections from which B. thuringiensis subsp. kurstaki HD1-like bacteria were isolated, and a measurement of the incidence and distribution of B. thuringiensis subsp. kurstaki HD1-like isolates, both in the environment and in the human population.

One of the difficulties in assessing the potential of B. thuringiensis subsp. kurstaki to cause human disease in previous health impact surveillance studies has been the inability to identify the Bacillus species recovered from samples as the specific isolate of B. thuringiensis applied in the spray. Without such identification, it is difficult to connect the specific isolate of B. thuringiensis applied in the spray to human exposure. The similarities among various B. thuringiensis strains, as well as among B. thuringiensis and certain strains of B. cereus, compound this problem. We report here on our efforts to identify B. thuringiensis subsp. kurstaki HD1-like bacteria in environmental and human samples collected before (prespray) and after (postspray) the three aerial applications of Foray 48B in Victoria, British Columbia.

	MATERIALS AND METHODS

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Background of the gypsy moth control program, definition of the spray zone, and spray dates. In the spring of 1999, the bacterial pest control product Foray 48B (Abbott Laboratories) was applied by aircraft (aerial spray) to selected areas of southern Vancouver Island to combat an infestation of the European gypsy moth, Lymantria dispar. The active ingredient of Foray 48B is B. thuringiensis subsp. kurstaki strain HD1. A total of 12,203 ha in the greater Victoria region were sprayed (Fig. 1). The spray zone included a mix of residential and rural areas and contained approximately 75,420 people. Sprays were applied in the early morning (between 5:00 and 7:00 a.m.) over three separate time periods: beginning on 8 May, 21 May, and 8 June 1999.

The number and type of samples. Two hundred fifty-six bulk air samples were taken during the aerial application of Foray 48B. These samples were taken by pulling air through 0.5-µm-pore-size Teflon filters mounted in 37-mm-diameter close-face cassettes, using constant-flow, battery-powered battery pumps (28). The pumps were calibrated to a flow of 2 liters/min ± 5% before and after sampling using a rotameter (Matheson). The pumps were operated for a period of 30 min during the aerial application of Foray 48B. The volume of air filtered was therefore approximately 60 liters.

Culturing bacteria from nasal swab samples. Bacteria were cultured from all nasal swabs at the Department of Medical Microbiology at the Victoria General Hospital, where they were cultured according to standard medical microbiological procedures (17) and screened for the presence of B. cereus group bacteria. Nasal swab isolates were considered as belonging to the B. cereus group if they demonstrated all six of the following characteristics: (i) large gram-positive bacilli, (ii) growth as flat or ground glass colonies that were hemolytic on sheep blood agar (Becton Dickinson Corp.), (iii) catalase positivity, (iv) motility, (v) lecithinase positivity on egg plates, and (vi) penicillin resistance (20). B. cereus group isolates were also examined microscopically for the presence of parasporal bodies indicative of B. thurigiensis.

Culturing bacteria from air samples. After the air samples were collected, they were delivered to our laboratory at the University of Victoria. The filter membranes were removed from the filter cassettes within a biological containment hood and placed facedown on nutrient agar (Difco Laboratories) plates without antibiotics. The plates were incubated 16 to 24 h at 28°C. Colonies were typically distributed uniformly on the filter membranes. The filter membranes were divided into equal sectors by drawing lines on the bottoms of the agar plates, and the total number of colonies was determined for each filter. Fifty colonies were picked randomly from the sectored plates and transferred to wells of a 96-well microtiter plate, each of which contained 200 µl of liquid Luria-Bertani broth (LB). The 96-well plates were incubated for 16 to 24 h at 28°C and then stored at 4°C until required for further use. A total of 3,701 bacterial colonies were isolated from the bulk air samples and then screened for the presence of B. thuringiensis subsp. kurstaki HD1-like bacteria (see below).

Bacterial strains. B. thuringiensis subsp. kurstaki HD1 was isolated from Foray 48B by plating the spray formulation on nutrient agar plates. The plates were incubated at 28°C for 16 to 24 h, and colonies were picked and transferred to 96-well plates. The plates were incubated for 16 to 24 h at 28°C and then stored at 4°C until required for further use. Defined specimens of B. thuringiensis subsp. kurstaki HD1, including several B. thuringiensis subsp. kurstaki HD1 strains that are negative for cry genes, as well as several different subspecies of B. thuringiensis (B. thuringiensis subsp. canadensis, B. thuringiensis subsp. entomocidus/subtoxicus, B. thuringiensis subsp. sotto/dendrolimus, and B. thuringiensis subsp. thuringiensis) were obtained from D. R. Zeigler at the Bacillus Genetic Stock Centre, Department of Biochemistry, Ohio State University (Table 1). Several control strains of B. cereus (strains 10876, 11778, 14579, and 21281) were obtained from A.-B. Kolsto at the Biotechnology Centre of Oslo, University of Oslo, Oslo, Norway.

RAPD analysis. All colonies of bacteria derived from nasal swabs were analyzed by use of random amplified polymorphic DNA (RAPD) analysis (30). DNAs used as the templates in the RAPD analyses were extracted from bacteria by incubation at 95°C for 10 min. A small amount of each bacterial colony was picked using a sterile loop and suspended in 0.2 ml of sterile, distilled, deionized water in a sterile 1.5-ml microcentrifuge tube. The microcentrifuge tubes were incubated at 100°C for 5 min and then placed on ice. One microliter of each supernatant was used in PCRs containing Ready to Go RAPD analysis beads (Amersham/Pharmacia Biotech). The random oligonucleotide primer 5'-GTTTCGCTCC-3' (RAPD primer 2; Amersham/Pharmacia Biotech) was chosen based on preliminary analyses using six strains of B. thuringiensis subsp. kurstaki (Table 1) and B. cereus (listed above). A second primer (RAPD primer 4, 5'-AAGAGCCCGT-3'; Amersham/Pharmacia Biotech) was used under the same conditions with the same strains. Reaction conditions consisted of an initial denaturation at 95°C for 4 min, followed by 45 cycles of denaturation at 94°C for 1 min, annealing at 36°C for 1 min, and extension at 72°C for 1 min. B. thuringiensis subsp. kurstaki HD1 isolated from Foray 48B was used as a positive control; B. cereus strain 10876 and Escherichia coli K-12 were used as negative controls.

cry1A PCR analysis. All colonies of bacteria derived from nasal swabs were subjected to PCR amplification to determine if the genes cry1Aa, cry1Ab, and cry1Ac were present. These genes are known to be characteristically, but not exclusively, present in B. thuringiensis subsp. kurstaki HD1 (5, 6, 7). PCR amplifications were performed in a final volume of 25 µl with 1× PCR buffer (50 mM KCl, 10 mM Tris-HCl, 1.5 mM MgCl₂), deoxynucleoside triphosphates (0.20 mM), Taq DNA polymerase (0.5 U; Gibco/BRL) and primers (1 µM each). The primer sequences and the sizes of expected amplification products are given in Tables 2 and 3, respectively. PCR conditions consisted of an initial denaturation at 95°C for 4 min, followed by cycles of denaturation at 94°C for 30 s, annealing at 50°C for 45 s, and extension at 72°C for 45 s. The amplified products were subjected to electrophoresis on 0.8% agarose gels at 100 V and visualized under UV light (wave length, 312 nm).

Dot blot DNA hybridization analysis. To confirm the results of RAPD analysis and cry gene PCR of nasal swabs, and to screen the large number of isolates from the air and water samples, we conducted dot blot analysis using standard DNA hybridization techniques (25). All bacteria from all samples received in our lab were inoculated into 0.2 ml of liquid nutrient broth aliquoted in sterile 96-well microtiter plates. Samples of B. cereus 10876 and B. thuringiensis subsp. kurstaki HD1 isolated from Foray 48B were cultured as negative and positive controls, respectively. The plates were incubated for 24 h at 28°C. Using a multichannel pipettor, 100 µl of each culture sample was transferred from the 96-well plates to plates consisting of 96 0.2-ml PCR tubes (Sarstedt). An equal volume of dot blot lysis buffer (0.8 M NaOH, 20 mM EDTA) was added to the tubes, resulting in final concentrations of 0.4 M NaOH and 10 mM EDTA. The tubes were capped tightly, placed in a thermal block, and incubated at 100°C for 10 min. Samples were placed on ice immediately and then filtered under vacuum through a nylon membrane (Zeta-Probe GT; Bio-Rad Corp.) using a Bio-Dot microfiltration manifold (Bio-Rad Corp.). The samples were washed with 0.5 ml of 0.4 M NaOH under vacuum and then rinsed in 2× SSC (1× SSC is 0.15 M NaCl plus 0.015 M sodium citrate). The DNA was fixed to the membrane by exposure to UV light (wavelength, 312 nm) for 3 min. cry1A gene PCR products were gel purified using a Wizard PCR product prep kit (Promega Corp.). The probes were labeled by incorporation of [-³²P]dCTP, using a random primer extension labeling kit with DNA polymerase I Klenow fragment (Gibco/BRL) according to the manufacturer's instructions. Hybridization was performed by following standard protocols (25). The hybridization patterns were visualized using a laser scanning imager (Storm PhosphorImager; Molecular Dynamics Corporation).

Statistical analyses. The incidences of bacteria with genetic patterns consistent with those of B. thuringiensis subsp. kurstaki HD1 isolated from human nasal swabs taken inside and outside the spray zone, both pre- and postspray, were analyzed for statistical differences using 2 by 3 (total number of isolates versus B. cereus group isolates versus B. thuringiensis subsp. kurstaki HD1-like positive isolates) chi-square analysis with 2 df. Chi-square values of >5.99 were considered statistically significant.

	RESULTS

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RAPD and cry Gene PCR analyses. RAPD analyses were performed using a number of B. thuringiensis HD1 isolates and five different subspecies of B. thuringiensis (Table 1), as well as four strains of B. cereus, using RAPD primers 2 and 4 (Amersham/Pharmacia Biotech). Both primer 2 (Fig. 2) and primer 4 (data not shown) were able to differentiate between B. thuringiensis subsp. kurstaki HD1 and the subspecies of B. thuringiensis tested, as well as between B. thuringiensis subsp. kurstaki HD1 and four B. cereus strains (data not shown). Under the conditions described above, RAPD reactions with primer 2 yielded characteristic products of approximately 1,000, 800, 600, and 400 bp with B. thuringiensis subsp. kurstaki HD1 (Fig. 2 and 3). Bands of approximately 2,800, 1,800, and 1,200 bp were observed rarely, occurring in only 6 out of the 208 (2.8%) samples analyzed. Variations in the amount of template in the RAPD PCRs may account for the observed variation in banding patterns (Ready to Go RAPD analysis bead instruction manual; Amersham/Pharmacia Biotech). Despite the rare appearance of these additional bands, RAPD analysis was able to distinguish among the six subspecies of B. thuringiensis tested (Fig. 2 and 3). While only B. cereus strain 10876 is shown in Fig. 2 and 3, RAPD analysis was able to distinguish B. thuringiensis subsp. kurstaki HD1 from the four B. cereus strains tested (data not shown).

View larger version (61K): [in this window] [in a new window]   FIG. 2.   RAPD analysis of several B. thuringiensis strains. Lanes: 1, B. cereus strain 10876; 2, B. thuringiensis subsp. kurstaki HD1 isolated from Foray 48B; 3, B. thuringiensis subsp. entomocidal/subtoxicus HD10; 4, B. thuringiensis subsp. kurstaki HD1 strain from the Bacillus Genetic Stock Centre; 5, E. coli K-12; 6, E. coli K-12; 7, B. thuringiensis subsp. kurstaki HD1 (cry-negative strain 4D7); 8, B. thuringiensis subsp. sotto/dendrolimus HD6; 9, B. thuringiensis subsp. kurstaki HD1 (cry-negative strain 4D8); 10, B. thuringiensis subsp. kurstaki HD1 (cry-negative strain 4D9); 11, B. thuringiensis subsp. entomocidal/subtoxicus HD10; 12, B. thuringiensis subsp. thuringiensis Bt1 (cry-negative strain); 13, B. thuringiensis subsp. israeliensis WHO 2013-9; 14, B. thuringiensis subsp. kurstaki HD1 (cry-negative strain 4D10); 15, B. thuringiensis subsp. kurstaki HD1 (cry-negative strain 4D11); 16, B. thuringiensis subsp. kurstaki HD1 (cry-negative strain 4D12); 17, B. thuringiensis subsp. canadensis HD224; 18, B. thuringiensis subsp. israeliensis Q2-81 pI. M, molecular size standard, 1-kb ladder. Lanes marked with a plus indicate samples with banding patterns that were consistent with those obtained with B. thuringiensis subsp. kurstaki strain HD1, and lanes marked with a minus indicate samples with banding patterns that were not consistent with those obtained with B. thuringiensis subsp. kurstaki strain HD1.

View larger version (67K): [in this window] [in a new window]   FIG. 3.   RAPD analysis of B. cereus group isolates from nasal swabs. Lanes: 1, negative control (c), B. cereus strain 10876; 2, positive control (+c), B. thuringiensis subsp. kurstaki HD1 isolated from Foray 48B; 3 to 7, banding patterns from nasal swabs that were negative for B. thuringiensis subsp. kurstaki HD1; 8 to 18, banding patterns for bacteria isolated from nasal swabs that were positive for B. thuringiensis subsp. kurstaki HD1-like isolates. A faint 600-bp band was detected in lane 10. Some higher-molecular-weight bands are also observed in lanes 8, 10, and 15. These differences may be due to different concentrations of the template used in the reaction. M, molecular size standard, 1-kb ladder. Lanes marked with a plus and lanes marked with a minus indicate samples with banding patterns that were and were not consistent with those obtained with B. thuringiensis subsp. kurstaki strain HD1, respectively.

View larger version (60K): [in this window] [in a new window]   FIG. 4.   cry gene PCR. Lanes 1 to 9, cry gene PCR products from B. cereus group isolates from nasal swab samples; lane 10, negative control (c), B. cereus strain 10876; lane 11, positive control (+c), B. thuringiensis subsp. kurstaki HD1 isolated from Foray 48B. B. thuringiensis subsp. kurstaki HD1-like isolates display the cry1Aa (1,500-bp), cry1Ab (858-bp), and cry1Ac (653-bp) PCR products. Only two of the three fragments are present in lanes 2 and 4, indicating that while these samples are B. thuringiensis, their cry gene banding patterns are not consistent with those of B. thuringiensis subsp. kurstaki HD1. RAPD analysis of these isolates also produced genetic profiles that differed from that of B. thuringiensis subsp. kurstaki HD1. M, molecular size standard, 1-kb ladder.

DNA hybridization. Typical hybridization results for bacteria isolated from nasal swabs and air samples are shown in Fig. 5. Dot blot membranes were hybridized, in turn, with ³²P-labeled probes that consisted of each of the three cry gene PCR products (the 1,500-, 858-, and 653-bp fragments) that had been previously gel purified. Figure 5 displays results obtained with the cry1Aa (1,500-bp) probe only. Results obtained with the cry1Ab (858-bp) and the cry1Ac (653-bp) probes are not shown but produced identical patterns of hybridization. Negative controls consisted of B. cereus strain 10876 (Fig. 5, lanes marked c), while the positive control consisted of B. thuringiensis subsp. kurstaki HD1 isolated from Foray 48B (Fig. 5, lanes marked +c). In some positions in the negative-control (B. cereus) row, we observed a faint signal, while in other positions in the same row, we observed no signal at all. Because all of the negative controls in the control row are derived from the same isolate of B. cereus, we may conclude that the faint hybridization detected is nonspecific background hybridization and therefore negative for B. thuringiensis subsp. kurstaki HD1-like isolates. Consequently, positions within the test rows with faint signals were considered negative.

View larger version (81K): [in this window] [in a new window]   FIG. 5.   Representative hybridization patterns of DNAs extracted from air and nasal swab samples. DNAs from individual bacterial colonies isolated from air samples (a total of 10,569) and from individual B. cereus group isolates (a total of 171) from nasal swabs were filtered onto nylon membranes and then hybridized with a 32P-labeled cry1Aa (1,500-bp) probe. Negative controls (c) consisted of B. cereus strain 10876; positive controls (+c) consisted of B. thuringiensis subsp. kurstaki HD1 isolated from Foray 48B. Dark spots are positive. Faint or light spots are considered nonspecific background hybridization and therefore negative. Membranes were stripped and then rehydridized with 32P-labeled cry1Ab (858-bp) and cry1Ac (653-bp) probes, which produced identical patterns of hybridization (not shown).

Identification of B. thuringiensis subsp. kurstaki HD1-like isolates in air samples. A combined total of 10,659 bacterial colonies isolated from the bulk air and Anderson plate samples were analyzed by DNA hybridization. Of the 3,701 colonies isolated from the bulk air samples, 2,978 (80.4%) showed a pattern of cry gene hybridization consistent with B. thuringiensis subsp. kurstaki HD1. Of the 6,958 colonies isolated from the Anderson plates, 6,124 (88.0%) showed a pattern consistent with B. thuringiensis subsp. kurstaki HD1. The distribution of air sampling locations is shown in Fig. 1. All samples were taken during the application of Foray 48B.

Identification of B. thuringiensis subsp. kurstaki HD1-like isolates in water samples. A total of 440 bacterial isolates, obtained from prespray (n = 114) and postspray (n = 326) water samples, were screened by DNA hybridization for the presence of B. thuringiensis subsp. kurstaki HD1-like bacteria (Table 4). Positive controls consisted of B. thuringiensis subsp. kurstaki HD1 isolates from Foray 48B, while the negative control consisted of B. cereus strain 10876. The concentration of all bacteria in pretreatment, prespray reservoir water was 390 CFU/ml for the Japan Gulch Reservoir and 260 CFU/ml for the Sooke Reservoir. The concentration of all bacteria in treated drinking water from the Japan Gulch Outlet was 5 CFU/ml. Of the 114 prespray sample colonies isolated, only one (0.87%), from the Sooke Reservoir, displayed a cry gene hybridization pattern consistent with that of B. thuringiensis subsp. kurstaki HD1. No B. thuringiensis subsp. kurstaki HD1-like isolates were identified in the prespray sample of treated drinking water collected at the Japan Gulch Outlet.

Identification of B. thuringiensis subsp. kurstaki HD1-like isolates in nasal swabs. Table 5 displays the total number of nasal swabs collected, the number of nasal swabs that were identified as B. cereus group, and the number of nasal swabs that displayed genetic patterns consistent with that of B. thuringiensis subsp. kurstaki HD1, collected prior to or after the first, second, and third applications of Foray 48B, both inside and outside the spray zone. Of the 171 B. cereus group isolates obtained from the nasal swabs, 131 (76.6%) had genetic patterns that were consistent with that of B. thuringiensis subsp. kurstaki HD1. While B. thuringiensis subsp. kurstaki HD1-like bacteria were detected in human nasal swabs taken from volunteers both inside and outside the spray zone prior to the first aerial application of Foray 48B, the differences were not significant statistically. After the first application of Foray 48B, however, the incidence of B. thuringiensis subsp. kurstaki HD1-like bacteria in human nasal swabs increased by a statistically significant amount (chi-square value = 9.37) in samples taken inside the spray zone but not in those taken outside the spray zone. By the time the spray program was completed, the incidences of B. thuringiensis subsp. kurstaki HD1-like bacteria in nasal swabs taken from both inside and outside the spray zone were significantly greater than the prespray rates. The combined counts for sprays 1, 2, and 3 had chi-square values of 8.95 and 22.29 for data collected inside and outside the spray-zone, respectively.

	DISCUSSION

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The gypsy moth control program in Victoria was considered a complete success, and the threat of an embargo on British Columbian lumber products was removed (29). Surveys conducted 1 year after aerial applications of Foray 48B failed to detect gypsy moth egg masses, and only four adult males were found in pheromone traps (History of gypsy moth infestations in British Columbia [2000], Ministry of Forests, Province of British Columbia website [htpp://www.for.gov.bc.ca/forsite/fhealth/history.html]).

There are very few cases recorded in the medical literature where B. thuringiensis has been accurately identified and has been directly associated with human infection. Epidemiological studies of populations exposed to B. thuringiensis subsp. kurstaki during insect control programs in urban areas have implicated B. thuringiensis subsp. kurstaki in clinical infections, but the methods used to identify the bacteria have not been conclusive and the isolates implicated have in almost all cases been considered contaminates (1, 2, 3, 16, 23; Noble et al., Abstr. 94th Gen. Meet. Am. Soc. Microbiol. 1994).

We employed and evaluated three molecular techniques to identify bacteria with genetic patterns consistent with those of B. thuringiensis subsp. kurstaki HD1 from other strains of B. thuringiensis, as well as from B. cereus, in environmental (air and water) and in human (nasal swabs) samples. While each technique provides certain advantages, they also have inherent limitations. The two major concerns about identification of B. thuringiensis subsp. kurstaki in this study were (i) the frequency at which bacterial isolates genetically consistent with B. thuringiensis subsp. kurstaki HD1 were not correctly identified (false negatives) and (ii) the frequency at which isolates that were not genetically consistent with B. thuringiensis subsp. kurstaki HD1 were incorrectly identified as B. thuringiensis subsp. kurstaki HD1-like bacteria (false positives).

While the probability of false positives generated by PCR and RAPD analyses is extremely low, these techniques are prone to generation of false negatives. PCRs can fail to generate amplification products for many reasons (10, 24). Moreover, the stability of the plasmids bearing the cry genes is also a factor. By subjecting the isolates to both analyses and cross-checking the results, we concluded that cry gene PCR was the least effective method of identifying B. thuringiensis subsp. kurstaki HD1-like bacteria, generating a high number of false negatives (32%).

B. thuringiensis subsp. kurstaki HD1 encodes three cry1 genes (cry1Aa, cry1Ab, and cry1Ac). Large-scale surveys of B. thuringiensis strains obtained from various locations around the world have found many isolates that encode these genes (5, 7, 11). The frequency and distribution of B. thuringiensis isolates that encode these three cry1A genes in British Columbia are unknown. Moreover, the 44-mDa plasmid that encodes the cry1Ab gene is known to be unstable (5, 7). Loss of this plasmid from a B. thuringiensis subsp. kurstaki isolate would alter the cry1A gene PCR profile, resulting in the exclusion of the isolate as a putative B. thuringiensis subsp. kurstaki HD1 isolate. In contrast, RAPD analysis, when performed with two primers, was very effective in differentiating B. thuringiensis subsp. kurstaki HD1-like bacteria from other varieties of B. thuringiensis, as well as from B. cereus, and generated a low level of false negatives.

In our study, cry gene PCR of two samples (Fig. 4, lanes 2 and 4) failed to detect the 858-bp cry1Ab fragment, suggesting that these samples were derived from non-B. thuringiensis subsp. kurstaki HD1-like bacteria or that these samples were examples of bacteria that had lost the 44-mDa plasmid and thus were negative for cry1Ab. Failure of the cry gene PCR in this case would be an example of false-negative results. RAPD analysis of these samples, however, also produced banding patterns that were different from that observed with B. thuringiensis subsp. kurstaki HD1. Thus, the RAPD analyses both confirmed the results of the cry gene PCR and indicated that these two isolates were not false negatives.

In contrast to PCR-based methods, DNA hybridization generates a very low frequency of false negatives but may generate false positives due to variations in the stringency of hybridization conditions. Hybridization, as expected, was the most effective method of identifying B. thuringiensis subsp. kurstaki HD1-like bacteria, with a rate of false negativity of only 2%. Hybridization is also much less costly and much more time-efficient and thus is the most appropriate technique for screening large numbers of samples. While the amount of DNA used in each of the three methods is crucial for the reproducibility of the results, the amount of DNA used in a hybridization reaction can vary to a greater extent without compromising the accuracy of detection.

Our data suggest that bacteria with genetic patterns consistent with those of B. thuringiensis subsp. kurstaki HD1 were present in both the environment and the human population of Victoria before aerial application of Foray 48B. B. thuringiensis subsp. kurstaki HD1-like bacteria were detected at low levels in the Victoria water reservoirs both prior to and after aerial applications of Foray 48B. The water reservoirs are located outside of the spray zone (approximately 2 km to the west of the western boundary of the spray zone) and in the upwind direction of the prevailing winds (southwest to northeast). Thus, the low levels of B. thuringiensis subsp. kurstaki HD1-like isolates detected in the drinking water, could have originated from other commercial products containing B. thuringiensis subsp. kurstaki HD1 or from naturally occurring populations with patterns similar to that of B. thuringiensis subsp. kurstaki HD1 and not from contamination as a consequence of the spray program. Nonetheless, B. thuringiensis subsp. kurstaki HD1-like bacteria were detected in drinking water after chloramination, and it is thus highly likely that residents of Victoria experience low levels of internal exposure to B. thuringiensis subsp. kurstaki HD1-like bacteria in drinking water.

B. thuringiensis subsp. kurstaki HD1-like bacteria were also isolated from nasal swabs taken from residents of Victoria, both inside and outside of the spray zone prior to the application of Foray 48B. The incidence of B. thuringiensis subsp. kurstaki HD1-like bacteria in the nares of the human population increased significantly within the spray zone, but not outside the spray zone, after the first aerial application of Foray 48B. By the time the spray program was completed, however, the incidence of B. thuringiensis subsp. kurstaki HD1-like isolates in the human population had increased significantly over prespray levels both inside and outside of the spray zone, suggesting widespread exposure to B. thuringiensis subsp. kurstaki HD1-like isolates, possibly due to the high levels of B. thuringiensis subsp. kurstaki HD1 spores remaining in the air after the spray applications.

Despite this exposure, however, the human health surveillance program failed to detect any correlation between the aerial application of B. thuringiensis subsp. kurstaki HD1 and short-term health effects in the general adult population, in emergency room visits, or in aggravation of asthma symptoms in children. While B. thuringiensis subsp. kurstaki HD1-like bacteria were detected in the nares of the human population, the available evidence suggests that its presence was transient, as clinical symptoms of active nasal-pharyngeal infection were not reported. Overall, the human health surveillance program did not detect any short-term change in health status that could be associated with the aerial application of Foray 48B.