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Michael K.

Rust, PhDa
Nancy C. Hinkle, PhDa
Marcella Waggonera
Norbert Mencke, DVM, PhDb
The Influence of Olaf Hansen, DVM, PhDb
Michael B. Vaughn, DVM, MSc

Imidacloprid on Adult a
Department of Entomology, University of California,
b
Riverside, California, USA
Bayer AG, BG-Animal Health, Monheim

Cat Flea Feeding c


Agricultural Center, D-51368 Leverkusen, Germany
Bayer Corporation, Agriculture Division, Animal Health,
Shawnee Mission, Kansas, USA

Introduction were exposed to cat hair treated with 0.025 and 0.01 ppm
The cat flea, Ctenocephalides felis felis (Bouché) is the primary imidacloprid.
ectoparasite of companion animals worldwide. Unlike many fleas,
the adult cat flea remains on the host where feeding, mating, and Methods and Materials
oviposition occur.1 Adult female cat fleas are aggressive feeders, Insects
consuming 13.6 µl of blood per day or the equivalent of 15 times The fleas tested were a susceptible UCR strain maintained in the
their body weight in blood.2 According to one study,3 66% of male laboratory.11 Larvae were fed an artificial diet containing 375 g
and 60% of female fleas fed within the first 5 minutes of contact and ground dog chow, 75 g dried beef blood, 50 g yeast, and 30-mesh
nearly 100% of the fleas had fed within 60 minutes. Typically, the sand (2/3 by volume). Adult cat fleas used in the study were
first blood meal lasted from 10 to 25 minutes. One consequence of obtained from rearing containers that contained eggs collected 18 to
this voracious feeding is that copious amounts of dried blood are 20 days earlier. The adults were lightly anesthetized with CO2 for
excreted, each flea producing about 0.77 mg of feces per day.4 The counting and placed into microcells.
host’s blood, especially the protein content, remains unaltered as it
passes through the flea digestive system.5 The primary source of Microcells
nutrition for larvae is dried fecal blood,6 and it is generally believed To confine the fleas to the host, fleas were placed in a microcell
that this is a special evolutionary adaptation allowing adult fleas to adapted from Osbrink and Rust12 (Figure 1A). The tops of snap cap
contribute to the development of larvae. plastic vials (2 cm diameter) were cut into 1.5 cm long sections. The
Most studies of the topical effects of insecticides applied to the cut end was glued with Devcon® 5 minute epoxy cement (ITW
skin of pet animals have focused on the efficacy or level of adult Performance Polymers, Riviera Beach, FL) to a disk of nylon
mortality produced. The effect on feeding behavior has been organdy fabric (2 cm diameter, 59 mesh). A 2 cm hole was cut in the
considered only recently. Cat fleas are primarily responsible for the center of two pieces (2.5 × 10 cm) of 3M Vetrap™ bandage tape
development of flea allergy dermatitis (FAD),7 and the inhibition of (3M Corp., St. Paul, MN). The two pieces were placed together and
adult cat flea feeding has tremendous implications for possibly the plastic vial was inserted in the hole so that the organdy mesh
preventing the initiation of FAD. For imidacloprid, recently was flush with the bandage. The vial was glued to the bandage with
published clinical trials have outlined the speed of flea kill 6, 12, 24, Devcon epoxy to hold it in place.
and 36 hours after spot-on treatment and subsequent reinfestations.8 About 50 mg of treated or untreated cat hair was placed inside
These results are in line with published results that fleas are affected the vial with 6 anesthetized adult fleas and the top of the vial was
within minutes after contact with imidacloprid-treated dog skin or closed with a plastic snap cap. The cat fleas were allowed 1 hour to
hair.9,10 It is generally assumed that the fleas that did not feed were recover from the CO2 in the microcell before testing. The organdy
killed by the treatments. membrane was placed on the shaved neck of a cat and secured with
This paper reports on the development of methods to determine an additional piece of Vetrap, which was wrapped at least twice
the effects of imidacloprid on adult cat fleas that would quantify the around the neck (Figure 1B). Studies have indicated that 97.2% of
amount of feeding, especially when exposed to low concentrations fleas are engorged within 1 hour of being placed on a host.3 After 18
of insecticide. Feeding was significantly reduced when adult cat fleas hours the microcell was removed and the number of live and dead fleas

Suppl Compend Contin Educ Pract Vet Vol. 23, No. 4(A), 2001 Second International Flea Control Symposium
Figure 1A Figure 1B
Figure 1—(A) Microcell used to hold fleas. (B) Putting microcell on a cat.

0.6 -

Absorbency (540 nm)


Absorbency (540 nm)

0.1 - y = –0.0057 + 0.0221x 0.5 -


r2 = 0.998
0.08 - 0.4 -
0.06 - 0.3 -
0.04 - 0.2 -
0.02 - 0.1 -
0- 0-
-

-
-

0 1 2 3 4 5 0 2 4 6
Hemoglobin (g/dl)
Flea Feces (mg/5 ml)

Figure 2—The curve of absorbency for the hemoglobin standard. Figure 3—The absorbency of flea feces in 5 mL of Drabkin’s solu-
The solid circles represent the absorbency values of serial dilutions tion.
of the standard compared with the projected linear equation.

were counted. The contents of the microcells and fleas were placed Adult Cat Flea Feeding
in 5 ml of Drabkin’s solution to dissolve all the blood. Six cat fleas were placed in microcells with untreated cat hair
and secured on the neck of shaved cats for 2, 4, 6, or 18 hours. Fleas
Blood Analyses were confined in the microcell for 1 hour before being placed on the
Qualitative determinations for hemoglobin were conducted with host to allow them to recover from the CO2. Each feeding period
Hemastix® reagent strips (Bayer Corporation, Elkhart, IN). Flea was replicated 4 times. The numbers of live and dead cat fleas were
fecal material and fleas were dissolved in about 5 ml of distilled counted. The microcells were placed in the freezer for at least 2
water. The Hemastix strip was dipped into the solution and hours to kill the adult fleas.
immediately removed. After 60 seconds, the color change of the To determine the amount of fecal material excreted by the fleas,
stick color (from orange to green) was compared to a chart that had the contents of the microcells were placed in 5 ml of Drabkin’s
categories for none, trace, small, moderate, or large. solution for 30 minutes. Samples were centrifuged for 15 minutes at
Quantitative determinations for the amount of hemoglobin in 3300 rpm to separate any fine particles or debris (Centrific®
individual fleas and fecal blood in the microcell were conducted Centrifuge, Fisher Scientific, Pittsburgh, PA). The samples were
with a diagnostic test that detects cyanmethemoglobin (Sigma placed in cuvettes and the absorbency determined with a
Diagnostics, St. Louis, MO).13 In this test, Drabkin’s solution reacts spectrophotometer (Beckman Model 35, Beckman Instruments,
with all forms of hemogloblin (except sulfhemoglobin, which is very Fullerton, CA). The amount of fecal material in the microcell was
rare in blood) to form cyanmethemoglobin. Cyanmethemoglobin is estimated from the linear regression for flea fecal material.
readily detected with either a narrow or broad band spectro- To determine the amount of blood in an adult flea, the fleas were
photometer at 540 nm. Hemoglobin standards were prepared (6, 12, individually crushed in 5 ml of Drabkin’s solution. The absorbency
and 18 g/dl) and the calibration curve of absorbency values versus was determined using the procedure above. The amount of
blood hemoglobin (g/dl) was linear. Lower serial dilutions were hemoglobin was estimated from the linear regression of the
prepared and compared with the expected values. hemoglobin standard.

TNAVC, January 2001 Suppl Compend Contin Educ Pract Vet Vol. 23, No. 4(A), 2001
0.9 - TABLE 1
0.8 -
0.7 - A
THE AMOUNT OF FEEDING OF ADULT
Absorbency

0.6 - CAT FLEAS CONFINED IN MICROCELLS


0.5 - ON THE NECK OF CATS
0.4 - B
0.3 - Flea Feces Hemoglobin/Flea
0.2 - Hours (mg/5 ml) (mg/dl)a
0.1 -
0-
-

-
2 1.51 2.34
460 500 550 600 650
Wavelength (nm) 4 4.96 3.20

6 10.33 1.84
Figure 4—The absorbency of the flea feces (A) and hemoglobin
standard (B) over the spectral range from 460 to 700 nm. 18 77.48 4.38

aTwo replicates of six cat fleas in each microcell.


To determine the effect of imidacloprid on feeding, fleas were
placed in a microcell along with treated cat hair. Samples (50 mg)
of cat hair were treated with 3 ml of 8 × 10-7 % and 1 × 10-8 %
imidacloprid (Bayer Animal Health, Monheim, Germany) in about 2.2 µg, consistent with findings that most blood rapidly passes
acetone (0.025 and 0.01 ppm). The hair was allowed to dry for 2 through the digestive system and remains unaltered.5
hours, then placed in the microcells along with 6 adult cat fleas. The Cat hair treated with 0.025 and 0.01 ppm imidacloprid
fleas were confined to the cat’s neck for 18 hours. After 18 hours, significantly decreased the amount of blood feeding by adult cat
the amount of flea feces and hemoglobin in the fleas was determined fleas on cats (Table 2). Small amounts of hemoglobin were still
by the methods described above. detected in adult fleas, suggesting that some feeding occurred, but
the amount of feces in the microcell was at the bottom limit of
Results and Discussion detection.
Chemical Analyses Fleas exposed to extremely high concentrations of insecticides
Analysis of the serial dilutions of the hemoglobin standard applied to the skin would not be expected to feed. As the residual
produced a linear equation (Figure 2; y = –0.0057 + 0.0221x, r2 = amount of insecticide available declines over time, the opportunity
0.998, t = 36.37, P < .001). When the standards were diluted to very for cat fleas to feed would increase. These predictions are supported
low concentrations and compared to the projected amounts by the findings of Franc and Cadiergues14,15 for pyrethroids such as
determined from the hemoglobin standard, they were not permethrin and deltamethrin and the combination of two
statistically different and fell within the 95% confidence limit (CL). organophosphates, dichlorvos and fenitrothion. In their study,
Validation of the technique to detect very low concentrations of topical applications of imidacloprid and fipronil did not prevent
hemoglobin was therefore confirmed. The bottom limits of detectable feeding in the fleas. Their observations were based on
detection in our tests were 0.303 g/dl hemoglobin (0.15 mg/5 ml). qualitative visual inspection of crushed fleas while our
Analysis of known quantities of flea feces produced a linear determinations were based on the amount of hemoglobin or flea
equation (Figure 3; y = –0.0060 + 0.0789x, r2 = 0.999, t = 116.5, P feces, a possible reason for the discrepancy between our findings and
< .001). The lowest sensitivity obtainable was 0.089 mg flea feces/5 theirs. In addition, only 1 hour elapsed from the time that the
ml. The amount of hemoglobin in the flea feces can be estimated imidacloprid was applied until they released fleas on the host. It is
only from the absorbency at 540 nm. The absorbency of the unlikely that the imidacloprid had an adequate opportunity to
dissolved fecal material was consistently 20% higher than spread before testing.
equivalent amounts of the hemoglobin standard over the spectral
range of 460 to 700 nm (Figure 4). Other components in the feces Summary
clearly resulted in higher absorbency than would be expected if the Applications of Advantage® (imidacloprid) to cats typically
feces were pure hemoglobin, consistent with other findings.15 provide about 20 to 30 ppm imidacloprid on the hairs at the day of
the treatment.16 Even after 30 days, hairs on treated cats had at least
Adult Cat Flea Feeding 1 ppm. These levels are substantially higher than the levels used in
The amount of fecal blood produced in the microcells increased our tests. We selected much lower doses so that we could determine
dramatically over 18 hours (Table 1) while the amount of if imidacloprid had a sublethal effect on feeding. Even the 0.01
hemoglobin in the fleas remained relatively constant (1.84 to 4.38 ppm–treated hair produced 66.7% mortality of fleas at 18 hours.
mg/dl). The maximum amount of hemoglobin in each flea was Lower doses of imidacloprid definitely inhibited feeding of adult

Suppl Compend Contin Educ Pract Vet Vol. 23, No. 4(A), 2001 Second International Flea Control Symposium
TABLE 2
THE AMOUNT OF FEEDING OF ADULT CAT FLEAS EXPOSED TO CAT
HAIR TREATED WITH 0.025 AND 0.01 PPM IMIDACLOPRID
% Dead at Avg. Fleaa Avg. hemoglobina
Treatment (ppm) at 18 hr feces (mg/5 ml) per flea (g/dL) (range)

0.025 100 0.238* 0.284 (0–0.303)*

None 0 3.684 n.s 0.575 (0–1.07) n.s.

0.01 66.7 0.320* 0.409 (0–0.52)*

None 0 6.385 n.s. 0.511 (0.349–0.801) n.s.

a,*indicates that 0.025 and 0.01 ppm were significantly different (P<0.05 Mann Whitney U test). Controls were not significantly different from one another.

cat fleas. Additional studies need to be conducted to determine the 8. Everett R, Cunningham J, Arther R, Bledsoe DL, Mencke N:
Comparative evaluation of the speed of flea kill of imidacloprid and
actual length of exposure required for the imidacloprid to inhibit
selamectin in dogs. Vet Ther 1:229–234, 2000.
feeding. These studies are an important beginning in our 9. Mehlhorn H, Mencke N, Hansen O: Effects of imidacloprid on adult
understanding of the role of insecticides such as imidacloprid in and larval stages of the cat flea Ctenocephalides felis after in vivo and in
vitro application: A light- and electron-microscopy study. Parasitol Res
preventing flea feeding and FAD.
85:625–637, 1999.
10. Mehlhorn H, Hansen O, Mencke N: Comparative study on the effects
References of three insecticides (fipronil, imidacloprid, selamectin) on the
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cat flea. Ann Rev Entomol 42: 451–473, 1997. A light and electron microscopic analysis of in vivo and in vitro
2. Dryden MW, Gaafar SM: Blood consumption by the cat flea, experiments. Parasitol Res 87:198–207, 2001.
Ctenocephalides felis (Siphonaptera: Pulicidae). J Med Entomol 28: 394- 11. Metzger ME, Rust MK: Egg production and emergence of adult cat fleas
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initiation and duration of feeding. J Med Entomol 37:634–636, 2000. Ctenocephalides felis felis (Siphonaptera: Pulicidae). J Med Entomol
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strategies of the act flea (Siphonaptera: Pulicidae). Florida Entomol 13. Kern WH Jr, Koehler PG, Patterson RS, Wadleigh RW: Spectro-
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excretion of host blood proteins in relation to larval nutrition. J Med 14. Franc M, Cadiergues MC: Activité anti-gorgement d’un shampooing à
Entomol 31:265–271, 1994. 0,07 % de deltaméthrine sur les puces du chien, Ctenocephalides felis. Rev
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Ctenocephalides felis felis (Bouché). Bull World Health Org 19:1126–1129, 1958. 15. Franc M, Cadiergues MC: Antifeeding effect of several insecticidal
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TNAVC, January 2001 Suppl Compend Contin Educ Pract Vet Vol. 23, No. 4(A), 2001

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