Advantage® International Flea Susceptibility

Monitoring Initiative—A 2001 Update

Byron L. Blagburn, PhDa Dennis E. Jacobs, BVMs, PhD,
Thomas Bach, DVMb FRCVS, FRCPathh
David L. Bledsoe, DVMc Heinz Mehlhorn, PhDi
Ian Denholm, PhDd Norbert Mencke, DVM, PhDb
Michael W. Dryden, DVM, PhDe Patricia A. Payne, DVM, PhDe
Olaf Hansen, DVM, PhDb Michael K. Rust, PhDf
Nancy C. Hinkle, PhDf Michael B. Vaughn, DVM, MSc
Terence Hopkins, BVSc (1st Class Hons.)g a
Auburn University, Alabama, USA
b
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 preva-
lent 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 ectopar-
asites 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 suscep-
tibility 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 devel-
oping and implementing an in vitro susceptibility monitoring assay
using imidacloprid as the test compound and four available labora-
tory strains of C. felis. The purpose of this article is to report progress
achieved by the research group in the year intervening between the
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 (Mon-
heim) strain. The Auburn University (AU) strain was already avail-
able at the test site. All had been maintained by serial propagation on
laboratory cats for variable lengths of time at the designated laborato-
ries 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 col-
lected 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 col-
lected 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

first and second Bayer International Flea Symposia. In this commu-
nication, we describe our in vitro C. felis larval bioassay and report
the susceptibilities of four established laboratory strains of C. felis to
imidacloprid.
all of the participating laboratories, specific acknowledgment should be giv-
en 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

Figure 1A Figure 1B
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.
1. Dissolve imidacloprid in acetone to achieve the following con-
centrations: 30, 15, 10, 5, 3, 1, 0.5, 0.1, and 0.05 ppm (mg/L).
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
rearing media in each of 9 tubes. Add 200 µl of acetone to the 10th
tube containing rearing media. Stir the mixture completely to dis-
perse the liquid (imidacloprid/acetone, acetone only) throughout
the rearing media. The final test concentrations of imidacloprid
achieved in the flea rearing media are 3, 1.5, 1.0, 0.5, 0.3, 0.1,
0.05, 0.01, 0.005, and 0.000 (control) ppm. Allow the acetone to
evaporate from the treated media by incubation in fume hood
overnight.
4. Transfer the contents of each plastic tube to individual covered
glass laboratory dishes.
5. On day 0 of the assay, collect 200 eggs of each laboratory strain
of C. felis. It is important that the eggs are collected at approxi-
mately the same time (e.g., within 2 hours).
6. Also on day 0, using a moistened sable brush, place a thin strip
of nontoxic, acid-free glue on the underside of the cover of each
laboratory dish (mentioned in step 4). Place each of the 200 eggs
from step 5 on the glue strip. Do this for all concentrations
including control. Place the laboratory dishes in an insectary or
incubator in conditions that will support flea development
(27 C; 75% to 85% relative humidity).The purpose of this pro-
˚ Kansas strains of C. felis were very similar in their responses to imi-
cedure is to allow larvae that hatch from the eggs to drop into
the media. In this way, the effects of the test compound on first-
stage larvae can be measured (Figure 1).
7. On day 5, enumerate the larvae that have successfully hatched
from eggs in each dish by examining eggs that remain on the glue
strip.
8. On study day 14, transfer all material in each of the laboratory
dishes into separate plastic vials with filter paper-vented lids and
return the vials to the incubator. At this stage in the assay, any sur-
viving larvae should have developed to the pupal (cocoon) stage.
9. On study day 28, enumerate all adult fleas in each of the plastic
vials.
TNAVC, January 2001
Determination of Treatment Responses
Treatment responses are determined by either count-
ing 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
adult fleas from the number of hatched larvae, which is
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
(acetone-treated media) assured that factors other than imidaclo-
prid were not affecting flea development.
Results and Discussion
Our initial experiences with the in vitro larval bioasssay suggest
that it will yield the desired susceptibility data with a minimum of
equipment, personnel, and time investment. Some initial problems
relating to difficulties in determining the numbers of viable larvae
that hatched from eggs and methods for separating and counting
adult fleas were resolved by refinement of assay techniques. Exam-
ples of dose responses of the four different laboratory strains of C.
felis in the larval bioassay conducted at Auburn University are giv-
en in Figures 2 and 3. Similar dose responses were obtained in the
laboratories of other team members, indicating that the larval
bioassay generally yields reproducible dose response data, regardless
of which laboratory personnel are conducting the assay. The read-
er is reminded that most components of the assay such as rearing
media, laboratory glass and plastic ware, insectary or incubator
environmental conditions, and specific assay procedures were stan-
dardized to eliminate as much as possible the variation these factors
can impose. We are satisfied that the standardized procedures
selected will result in continuing reproducible data as we enter into
the field monitoring phase to follow (see below). The Auburn and
dacloprid (Figures 2A, 2B, and 3). LD50 (0.46 and 0.44 ppm) and
LD 95 (0.95 and 0.80 ppm) dosage rates were somewhat higher than
those observed for the Monheim (Figure 2C) and UCR (Figure 2D)
strains, which also were similar to each other. Ranges of LD50 dose
rates for the four strains were considerably less than ranges
observed for the LD95 dose rates (Figure 3). These results are con-
sistent with those observed for other insects and other insecticides
evaluated in similarly conducted dose-response studies.
Having established a reproducible and user-friendly bioassay, we
feel that we are now prepared to enter the field flea susceptibility
monitoring phase of the initiative. During this phase it is our inten-
tion to collect and evaluate the susceptibility of numerous field iso-
Suppl Compend Contin Educ Pract Vet Vol. 23, No. 4(A), 2001
 Auburn Strain KSU Strain
10 10
LD10 = 0.260 9 LD10 = 0.279 9
LD50 = 0.459 8 LD50 = 0.443 8
LD95 = 0.950

Probit of Response
Probit of Response
7 LD95 = 0.801 7

6 6

5 5

4 4

3 3

2 2

1 1

0 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 UCR Strain

10 10

9 9
LD10 = 0.239 LD10 = 0.231
LD50 = 0.353 8 LD50 = 0.316 8
Probit of Response

Probit of Response
LD95 = 0.582 7 LD95 = 0.472 7

6 6

5 5

4 4

3 3

2 2
1 1

0 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 C. felis collected from client animals in collaboration with practic-
.80
ing veterinarians. It remains our intention at this stage in the ini-
0.8 tiative to sample isolates randomly without any bias placed on a flea
(ppm) mg/L

.58 Au bur n strain’s response to prior treatment with insecticides. Traditionally,

0.6 K SU
.47 discriminating doses are placed at multiples of the LD95 (e.g., 3 times
.46 .44 M on he im
U CR the mean or median LD95 value for susceptible laboratory and field
0.4 .35
.32
isolates.) The discriminating dose will be selected following thor-
0.2 ough analysis of all dose-response data from laboratory or available
field flea isolates.
0
LD 50 LD 95
Acknowledgments
The authors gratefully acknowledge Ms. Mel Hutchinson, Ms.
Figure 3—Summary of susceptibility of four laboratory flea strains
to imidacloprid. Tracey Land, Ms. Marcella Waggoner, Ms. Jody Hampton-Beesley,
and Ms. Vicki Smith for their invaluable assistance in the develop-
ment and implementation of this initiative.
lates of C. felis from several geographic regions in the United States,
Europe, and perhaps other parts of the world. Prior to initiating the References
monitoring phase, we must first select a discriminating imidacloprid 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
 ance. Parasitol Today 9(5):174–179, 1993.
2. Brown AW, Pal R: Insecticide Resistance in Arthropods. WHO Mono-
graph 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.
TNAVC, January 2001
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.
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