ADVANTAGE(Flea Control)
Content
Advantage® International Flea Susceptibility Monitoring Initiative—A 2001 Update
Byron L. Blagburn, PhDa Thomas Bach, DVMb David L. Bledsoe, DVMc Ian Denholm, PhDd Michael W. Dryden, DVM, PhDe Olaf Hansen, DVM, PhDb Nancy C. Hinkle, PhDf Terence Hopkins, BVSc (1st Class Hons.)g Dennis E. Jacobs, BVMs, PhD, FRCVS, FRCPathh Heinz Mehlhorn, PhDi Norbert Mencke, DVM, PhDb Patricia A. Payne, DVM, PhDe Michael K. Rust, PhDf Michael B. Vaughn, DVM, MSc
b
a Auburn University, Alabama, USA Bayer AG, BG-Animal Health, Monheim Agricultural Center, D-51368 Leverkusen, Germany c Bayer Corporation, Agriculture Division, Animal Health, Shawnee Mission, Kansas, USA d IACR-Rothamsted, UK e Kansas State University, Manhattan, Kansas, USA f University of California, Riverside, California, USA g Bayer Australia, Beenleign, Queensland, Australia h The Royal Veterinary College (University of London), North Mymms, UK i Heinrich-Heine University, Düsseldorf, Germany
E
ctoparasiticide resistance represents an increasingly prevalent problem facing industry and agriculture. To date, more than 500 species of insects, mites, or ticks are resistant to one or more insecticides.1 Resistance is not restricted to a single or even a few chemical classes of insecticides but has been demonstrated for many inorganic and synthetic organic compounds.2 The majority of ectoparasites with demonstrated resistance to pesticides are crop pests. However, approximately 40% of resistant species are ectoparasites of humans or other animals.1 The cat flea, Ctenocephalides felis (Bouché 1835) is the flea most commonly observed on pets and other animals in both the United States and Europe.3 According to the World Health Organization, C. felis is resistant to more insecticides in more chemical categories than has been reported for any other species of flea.4 Despite the known capability of the cat flea to develop resistance to commonly applied insecticides, no effort is being made to monitor the susceptibility of C. felis to currently marketed flea insecticides. In response to the need for such a monitoring initiative, the authors, with support from Bayer Animal Health, set about developing and implementing an in vitro susceptibility monitoring assay using imidacloprid as the test compound and four available laboratory strains of C. felis. The purpose of this article is to report progress achieved by the research group in the year intervening between the first and second Bayer International Flea Symposia. In this communication, we describe our in vitro C. felis larval bioassay and report the susceptibilities of four established laboratory strains of C. felis to imidacloprid.
Methods
Laboratory Strains of C. felis
The following three laboratory strains of C. felis were shipped as cocoons to Auburn University by overnight courier: The University of California at Riverside (UCR) strain; the Kansas State University (KSU) strain; and the Bayer Laboratories, Monheim, Germany (Monheim) strain. The Auburn University (AU) strain was already available at the test site. All had been maintained by serial propagation on laboratory cats for variable lengths of time at the designated laboratories prior to their propagation and maintenance at the Auburn test site. Adult fleas that emerged from cocoons were immediately placed on commercially obtained laboratory cats that were known to be free of fleas. Cats were housed individually in stainless steel cages fitted with grated floor walks that facilitated collection of flea eggs. Eggs collected from cats infested with each laboratory strain were reared to adult fleas. These adults were again used to reinfest the same cat. This procedure was repeated until a sufficient numbers of eggs could be collected to permit conduct of the larval bioasssay described below.
Larval Bioassay
Susceptibility of the laboratory strains of C. felis to imidacloprid was determined using the following procedurej:
jAlthough this procedure was developed and modified as a result of efforts in all of the participating laboratories, specific acknowledgment should be given to Dr. Olaf Hansen at the Monheim laboratory and Dr. Michael Rust at the University of California laboratories for their efforts in identifying and implementing key methodologies used in the assay. Imidacloprid was obtained from Bayer Laboratories, Monheim, Germany.
Suppl Compend Contin Educ Pract Vet Vol. 23, No. 4(A), 2001
Second International Flea Control Symposium
Determination of Treatment Responses
Treatment responses are determined by either counting the numbers of dead larvae or counting the adult fleas contained in the media at each dosage level. In the former case, the treatment response would be the ratio of the number of dead larvae to the number of larvae that hatched successfully. In the latter case, the treatment response would be calculated by subtracting the number of Figure 1A Figure 1B adult fleas from the number of hatched larvae, which is Figure 1—(A) Hatched eggs of C. felis and debris retained on the glue strip. then divided by the number of hatched larvae. Regardless, (B) Closeup view of one hatched egg. the treatment response at each of the concentrations of imidacloprid was converted to its corresponding probit5 and graphed against the log of the dose to linearize the data. A probit line was approximated for each data set. The controls 1. Dissolve imidacloprid in acetone to achieve the following con(acetone-treated media) assured that factors other than imidaclocentrations: 30, 15, 10, 5, 3, 1, 0.5, 0.1, and 0.05 ppm (mg/L). prid were not affecting flea development. 2. Add 2 g of flea rearing media (375 g ground dry dog ration, 75 g dried bovine blood, 2 g inactive baker’s yeast [1/3 by volume], and sand [2/3 by volume]) to each of 10 individual plastic tubes. 3. Add 200 µl of each concentration of imidacloprid to the 2 g of Our initial experiences with the in vitro larval bioasssay suggest rearing media in each of 9 tubes. Add 200 µl of acetone to the 10th that it will yield the desired susceptibility data with a minimum of tube containing rearing media. Stir the mixture completely to disequipment, personnel, and time investment. Some initial problems perse the liquid (imidacloprid/acetone, acetone only) throughout relating to difficulties in determining the numbers of viable larvae the rearing media. The final test concentrations of imidacloprid that hatched from eggs and methods for separating and counting achieved in the flea rearing media are 3, 1.5, 1.0, 0.5, 0.3, 0.1, adult fleas were resolved by refinement of assay techniques. Exam0.05, 0.01, 0.005, and 0.000 (control) ppm. Allow the acetone to ples of dose responses of the four different laboratory strains of C. evaporate from the treated media by incubation in fume hood felis in the larval bioassay conducted at Auburn University are givovernight. en in Figures 2 and 3. Similar dose responses were obtained in the 4. Transfer the contents of each plastic tube to individual covered laboratories of other team members, indicating that the larval glass laboratory dishes. bioassay generally yields reproducible dose response data, regardless 5. On day 0 of the assay, collect 200 eggs of each laboratory strain of which laboratory personnel are conducting the assay. The readof C. felis. It is important that the eggs are collected at approxier is reminded that most components of the assay such as rearing mately the same time (e.g., within 2 hours). media, laboratory glass and plastic ware, insectary or incubator 6. Also on day 0, using a moistened sable brush, place a thin strip environmental conditions, and specific assay procedures were stanof nontoxic, acid-free glue on the underside of the cover of each dardized to eliminate as much as possible the variation these factors laboratory dish (mentioned in step 4). Place each of the 200 eggs can impose. We are satisfied that the standardized procedures from step 5 on the glue strip. Do this for all concentrations selected will result in continuing reproducible data as we enter into including control. Place the laboratory dishes in an insectary or the field monitoring phase to follow (see below). The Auburn and incubator in conditions that will support flea development Kansas strains of C. felis were very similar in their responses to imi˚ (27 C; 75% to 85% relative humidity).The purpose of this prodacloprid (Figures 2A, 2B, and 3). LD50 (0.46 and 0.44 ppm) and cedure is to allow larvae that hatch from the eggs to drop into LD95 (0.95 and 0.80 ppm) dosage rates were somewhat higher than the media. In this way, the effects of the test compound on firstthose observed for the Monheim (Figure 2C) and UCR (Figure 2D) stage larvae can be measured (Figure 1). strains, which also were similar to each other. Ranges of LD50 dose 7. On day 5, enumerate the larvae that have successfully hatched rates for the four strains were considerably less than ranges from eggs in each dish by examining eggs that remain on the glue observed for the LD95 dose rates (Figure 3). These results are constrip. sistent with those observed for other insects and other insecticides 8. On study day 14, transfer all material in each of the laboratory evaluated in similarly conducted dose-response studies. dishes into separate plastic vials with filter paper-vented lids and Having established a reproducible and user-friendly bioassay, we return the vials to the incubator. At this stage in the assay, any surfeel that we are now prepared to enter the field flea susceptibility viving larvae should have developed to the pupal (cocoon) stage. monitoring phase of the initiative. During this phase it is our inten9. On study day 28, enumerate all adult fleas in each of the plastic tion to collect and evaluate the susceptibility of numerous field isovials.
Results and Discussion
TNAVC, January 2001
Suppl Compend Contin Educ Pract Vet Vol. 23, No. 4(A), 2001
Auburn Strain
10
KSU Strain
10
7 6 5 4 3 2 1 0
Probit of Response
7 6 5 4 3 2 1 0
–2.5
–2
–1.5
–1
–0.5
0
0.5
1
–2.5
–2
–1.5
–1
–0.5
0
0.5
1
Log of Dose
Log of Dose
Figure 2A
Figure 2B
Monheim Strain
10
UCR Strain
10 9 8 Probit of Response 7 6 5 4 3 2 1 0
7 6 5 4 3 2 1 0
–2.5
–2
–1.5
–1
–0.5
0
0.5
1
–2.5
–2
–1.5
–1
–0.5
0
0.5
1
Log of Dose
Log of Dose
Figure 2C Figure 2D Figure 2—Efficacy of imidacloprid against four laboratory strains of Ctenocephalides felis in the larval bioassay.
dose. We will use the discriminating dose to screen field isolates of
1
.95 .80 .58 .46 .44 .47 .35 .32
(ppm) mg/L
0.8 0.6 0.4 0.2 0 LD 50
Au bur n K SU M on he im U CR
C. felis collected from client animals in collaboration with practicing veterinarians. It remains our intention at this stage in the initiative to sample isolates randomly without any bias placed on a flea strain’s response to prior treatment with insecticides. Traditionally, discriminating doses are placed at multiples of the LD95 (e.g., 3 times the mean or median LD95 value for susceptible laboratory and field isolates.) The discriminating dose will be selected following thorough analysis of all dose-response data from laboratory or available field flea isolates.
LD 95
Acknowledgments
The authors gratefully acknowledge Ms. Mel Hutchinson, Ms. Tracey Land, Ms. Marcella Waggoner, Ms. Jody Hampton-Beesley, and Ms. Vicki Smith for their invaluable assistance in the development and implementation of this initiative.
Figure 3—Summary of susceptibility of four laboratory flea strains to imidacloprid.
lates of C. felis from several geographic regions in the United States, Europe, and perhaps other parts of the world. Prior to initiating the monitoring phase, we must first select a discriminating imidacloprid
References
1. Roush RT: Occurrence, genetics and management of insecticide resist-
Suppl Compend Contin Educ Pract Vet Vol. 23, No. 4(A), 2001
Second International Flea Control Symposium
Probit of Response
LD10 = 0.239 LD50 = 0.353 LD95 = 0.582
LD10 = 0.231 LD50 = 0.316 LD95 = 0.472
9 8
Probit of Response
LD10 = 0.260 LD50 = 0.459 LD95 = 0.950
9 8
LD10 = 0.279 LD50 = 0.443 LD95 = 0.801
9 8
ance. Parasitol Today 9(5):174–179, 1993. 2. Brown AW, Pal R: Insecticide Resistance in Arthropods. WHO Monograph Series No. 38. Geneva, World Health Organization, 1971. 3. Rust MK, Dryden MD: The biology, ecology, and management of the cat flea. Annu Rev Entomol 42:451–473, 1997.
4. Rust MK: Insecticide resistance in fleas, in Knapp FW (ed): Proceedings of the International Symposium on Ectoparasites of Pets. April 4–6, 1993. Lexington, KY, University of Kentucky, 1993. 5. Finney DJ: Probit Analysis. Cambridge, Cambridge University Press, 1971.
TNAVC, January 2001
Suppl Compend Contin Educ Pract Vet Vol. 23, No. 4(A), 2001
Byron L. Blagburn, PhDa Thomas Bach, DVMb David L. Bledsoe, DVMc Ian Denholm, PhDd Michael W. Dryden, DVM, PhDe Olaf Hansen, DVM, PhDb Nancy C. Hinkle, PhDf Terence Hopkins, BVSc (1st Class Hons.)g Dennis E. Jacobs, BVMs, PhD, FRCVS, FRCPathh Heinz Mehlhorn, PhDi Norbert Mencke, DVM, PhDb Patricia A. Payne, DVM, PhDe Michael K. Rust, PhDf Michael B. Vaughn, DVM, MSc
b
a Auburn University, Alabama, USA Bayer AG, BG-Animal Health, Monheim Agricultural Center, D-51368 Leverkusen, Germany c Bayer Corporation, Agriculture Division, Animal Health, Shawnee Mission, Kansas, USA d IACR-Rothamsted, UK e Kansas State University, Manhattan, Kansas, USA f University of California, Riverside, California, USA g Bayer Australia, Beenleign, Queensland, Australia h The Royal Veterinary College (University of London), North Mymms, UK i Heinrich-Heine University, Düsseldorf, Germany
E
ctoparasiticide resistance represents an increasingly prevalent problem facing industry and agriculture. To date, more than 500 species of insects, mites, or ticks are resistant to one or more insecticides.1 Resistance is not restricted to a single or even a few chemical classes of insecticides but has been demonstrated for many inorganic and synthetic organic compounds.2 The majority of ectoparasites with demonstrated resistance to pesticides are crop pests. However, approximately 40% of resistant species are ectoparasites of humans or other animals.1 The cat flea, Ctenocephalides felis (Bouché 1835) is the flea most commonly observed on pets and other animals in both the United States and Europe.3 According to the World Health Organization, C. felis is resistant to more insecticides in more chemical categories than has been reported for any other species of flea.4 Despite the known capability of the cat flea to develop resistance to commonly applied insecticides, no effort is being made to monitor the susceptibility of C. felis to currently marketed flea insecticides. In response to the need for such a monitoring initiative, the authors, with support from Bayer Animal Health, set about developing and implementing an in vitro susceptibility monitoring assay using imidacloprid as the test compound and four available laboratory strains of C. felis. The purpose of this article is to report progress achieved by the research group in the year intervening between the first and second Bayer International Flea Symposia. In this communication, we describe our in vitro C. felis larval bioassay and report the susceptibilities of four established laboratory strains of C. felis to imidacloprid.
Methods
Laboratory Strains of C. felis
The following three laboratory strains of C. felis were shipped as cocoons to Auburn University by overnight courier: The University of California at Riverside (UCR) strain; the Kansas State University (KSU) strain; and the Bayer Laboratories, Monheim, Germany (Monheim) strain. The Auburn University (AU) strain was already available at the test site. All had been maintained by serial propagation on laboratory cats for variable lengths of time at the designated laboratories prior to their propagation and maintenance at the Auburn test site. Adult fleas that emerged from cocoons were immediately placed on commercially obtained laboratory cats that were known to be free of fleas. Cats were housed individually in stainless steel cages fitted with grated floor walks that facilitated collection of flea eggs. Eggs collected from cats infested with each laboratory strain were reared to adult fleas. These adults were again used to reinfest the same cat. This procedure was repeated until a sufficient numbers of eggs could be collected to permit conduct of the larval bioasssay described below.
Larval Bioassay
Susceptibility of the laboratory strains of C. felis to imidacloprid was determined using the following procedurej:
jAlthough this procedure was developed and modified as a result of efforts in all of the participating laboratories, specific acknowledgment should be given to Dr. Olaf Hansen at the Monheim laboratory and Dr. Michael Rust at the University of California laboratories for their efforts in identifying and implementing key methodologies used in the assay. Imidacloprid was obtained from Bayer Laboratories, Monheim, Germany.
Suppl Compend Contin Educ Pract Vet Vol. 23, No. 4(A), 2001
Second International Flea Control Symposium
Determination of Treatment Responses
Treatment responses are determined by either counting the numbers of dead larvae or counting the adult fleas contained in the media at each dosage level. In the former case, the treatment response would be the ratio of the number of dead larvae to the number of larvae that hatched successfully. In the latter case, the treatment response would be calculated by subtracting the number of Figure 1A Figure 1B adult fleas from the number of hatched larvae, which is Figure 1—(A) Hatched eggs of C. felis and debris retained on the glue strip. then divided by the number of hatched larvae. Regardless, (B) Closeup view of one hatched egg. the treatment response at each of the concentrations of imidacloprid was converted to its corresponding probit5 and graphed against the log of the dose to linearize the data. A probit line was approximated for each data set. The controls 1. Dissolve imidacloprid in acetone to achieve the following con(acetone-treated media) assured that factors other than imidaclocentrations: 30, 15, 10, 5, 3, 1, 0.5, 0.1, and 0.05 ppm (mg/L). prid were not affecting flea development. 2. Add 2 g of flea rearing media (375 g ground dry dog ration, 75 g dried bovine blood, 2 g inactive baker’s yeast [1/3 by volume], and sand [2/3 by volume]) to each of 10 individual plastic tubes. 3. Add 200 µl of each concentration of imidacloprid to the 2 g of Our initial experiences with the in vitro larval bioasssay suggest rearing media in each of 9 tubes. Add 200 µl of acetone to the 10th that it will yield the desired susceptibility data with a minimum of tube containing rearing media. Stir the mixture completely to disequipment, personnel, and time investment. Some initial problems perse the liquid (imidacloprid/acetone, acetone only) throughout relating to difficulties in determining the numbers of viable larvae the rearing media. The final test concentrations of imidacloprid that hatched from eggs and methods for separating and counting achieved in the flea rearing media are 3, 1.5, 1.0, 0.5, 0.3, 0.1, adult fleas were resolved by refinement of assay techniques. Exam0.05, 0.01, 0.005, and 0.000 (control) ppm. Allow the acetone to ples of dose responses of the four different laboratory strains of C. evaporate from the treated media by incubation in fume hood felis in the larval bioassay conducted at Auburn University are givovernight. en in Figures 2 and 3. Similar dose responses were obtained in the 4. Transfer the contents of each plastic tube to individual covered laboratories of other team members, indicating that the larval glass laboratory dishes. bioassay generally yields reproducible dose response data, regardless 5. On day 0 of the assay, collect 200 eggs of each laboratory strain of which laboratory personnel are conducting the assay. The readof C. felis. It is important that the eggs are collected at approxier is reminded that most components of the assay such as rearing mately the same time (e.g., within 2 hours). media, laboratory glass and plastic ware, insectary or incubator 6. Also on day 0, using a moistened sable brush, place a thin strip environmental conditions, and specific assay procedures were stanof nontoxic, acid-free glue on the underside of the cover of each dardized to eliminate as much as possible the variation these factors laboratory dish (mentioned in step 4). Place each of the 200 eggs can impose. We are satisfied that the standardized procedures from step 5 on the glue strip. Do this for all concentrations selected will result in continuing reproducible data as we enter into including control. Place the laboratory dishes in an insectary or the field monitoring phase to follow (see below). The Auburn and incubator in conditions that will support flea development Kansas strains of C. felis were very similar in their responses to imi˚ (27 C; 75% to 85% relative humidity).The purpose of this prodacloprid (Figures 2A, 2B, and 3). LD50 (0.46 and 0.44 ppm) and cedure is to allow larvae that hatch from the eggs to drop into LD95 (0.95 and 0.80 ppm) dosage rates were somewhat higher than the media. In this way, the effects of the test compound on firstthose observed for the Monheim (Figure 2C) and UCR (Figure 2D) stage larvae can be measured (Figure 1). strains, which also were similar to each other. Ranges of LD50 dose 7. On day 5, enumerate the larvae that have successfully hatched rates for the four strains were considerably less than ranges from eggs in each dish by examining eggs that remain on the glue observed for the LD95 dose rates (Figure 3). These results are constrip. sistent with those observed for other insects and other insecticides 8. On study day 14, transfer all material in each of the laboratory evaluated in similarly conducted dose-response studies. dishes into separate plastic vials with filter paper-vented lids and Having established a reproducible and user-friendly bioassay, we return the vials to the incubator. At this stage in the assay, any surfeel that we are now prepared to enter the field flea susceptibility viving larvae should have developed to the pupal (cocoon) stage. monitoring phase of the initiative. During this phase it is our inten9. On study day 28, enumerate all adult fleas in each of the plastic tion to collect and evaluate the susceptibility of numerous field isovials.
Results and Discussion
TNAVC, January 2001
Suppl Compend Contin Educ Pract Vet Vol. 23, No. 4(A), 2001
Auburn Strain
10
KSU Strain
10
7 6 5 4 3 2 1 0
Probit of Response
7 6 5 4 3 2 1 0
–2.5
–2
–1.5
–1
–0.5
0
0.5
1
–2.5
–2
–1.5
–1
–0.5
0
0.5
1
Log of Dose
Log of Dose
Figure 2A
Figure 2B
Monheim Strain
10
UCR Strain
10 9 8 Probit of Response 7 6 5 4 3 2 1 0
7 6 5 4 3 2 1 0
–2.5
–2
–1.5
–1
–0.5
0
0.5
1
–2.5
–2
–1.5
–1
–0.5
0
0.5
1
Log of Dose
Log of Dose
Figure 2C Figure 2D Figure 2—Efficacy of imidacloprid against four laboratory strains of Ctenocephalides felis in the larval bioassay.
dose. We will use the discriminating dose to screen field isolates of
1
.95 .80 .58 .46 .44 .47 .35 .32
(ppm) mg/L
0.8 0.6 0.4 0.2 0 LD 50
Au bur n K SU M on he im U CR
C. felis collected from client animals in collaboration with practicing veterinarians. It remains our intention at this stage in the initiative to sample isolates randomly without any bias placed on a flea strain’s response to prior treatment with insecticides. Traditionally, discriminating doses are placed at multiples of the LD95 (e.g., 3 times the mean or median LD95 value for susceptible laboratory and field isolates.) The discriminating dose will be selected following thorough analysis of all dose-response data from laboratory or available field flea isolates.
LD 95
Acknowledgments
The authors gratefully acknowledge Ms. Mel Hutchinson, Ms. Tracey Land, Ms. Marcella Waggoner, Ms. Jody Hampton-Beesley, and Ms. Vicki Smith for their invaluable assistance in the development and implementation of this initiative.
Figure 3—Summary of susceptibility of four laboratory flea strains to imidacloprid.
lates of C. felis from several geographic regions in the United States, Europe, and perhaps other parts of the world. Prior to initiating the monitoring phase, we must first select a discriminating imidacloprid
References
1. Roush RT: Occurrence, genetics and management of insecticide resist-
Suppl Compend Contin Educ Pract Vet Vol. 23, No. 4(A), 2001
Second International Flea Control Symposium
Probit of Response
LD10 = 0.239 LD50 = 0.353 LD95 = 0.582
LD10 = 0.231 LD50 = 0.316 LD95 = 0.472
9 8
Probit of Response
LD10 = 0.260 LD50 = 0.459 LD95 = 0.950
9 8
LD10 = 0.279 LD50 = 0.443 LD95 = 0.801
9 8
ance. Parasitol Today 9(5):174–179, 1993. 2. Brown AW, Pal R: Insecticide Resistance in Arthropods. WHO Monograph Series No. 38. Geneva, World Health Organization, 1971. 3. Rust MK, Dryden MD: The biology, ecology, and management of the cat flea. Annu Rev Entomol 42:451–473, 1997.
4. Rust MK: Insecticide resistance in fleas, in Knapp FW (ed): Proceedings of the International Symposium on Ectoparasites of Pets. April 4–6, 1993. Lexington, KY, University of Kentucky, 1993. 5. Finney DJ: Probit Analysis. Cambridge, Cambridge University Press, 1971.
TNAVC, January 2001
Suppl Compend Contin Educ Pract Vet Vol. 23, No. 4(A), 2001
