Veterinary Parasitology 116 (2003) 45–50
Comparison of the activity of selamectin, fipronil,
and imidacloprid against flea larvae
(Ctenocephalides felis felis) in vitro
T.L. McTier a,∗, N.A. Evans b, M. Martin-Short c, K. Gration c
a Pfizer Animal Health Group, Veterinary Medicine Research and Development,
Eastern Point Road, Mail Stop 8200-40, Groton, CT 06340, USA
b Pfizer Animal Health Group, Technical Product Development, New York, NY, USA
c Pfizer Animal Health Group, Veterinary Medicine Research and Development,
Sandwich, Kent CT13 9NJ, Kent, UK
Received 23 October 2002; received in revised form 14 April 2003; accepted 15 April 2003
Abstract
The activity of selamectin, fipronil and imidacloprid against larval cat fleas (Ctenocephalides
felis felis) was evaluated in an in vitro potency assay system. One hundred microliters of each
compound at various concentrations in acetone were added to glass vials (1.5 by 3 cm) to which
had been previously added 20 mg of sand and 10 mg of flea feces. Vials were then ball milled to
allow the acetone to evaporate. Selamectin and fipronil were tested at 0.001, 0.003, 0.005, 0.01,
0.03, 0.05, 0.11, 0.3, and 0.5g of active compound per tube. Imidacloprid was tested at 0.01,
0.03, 0.05, 0.1, 0.3, 0.5, 1.0, 3.0, and 5.0g of active compound per tube. Thirty first instar C. felis
larvae were added to each vial. The number of larvae remaining alive in each vial was determined
once daily for 72 h. With selamectin, reductions of ≥93.5% were achieved at 24 h after exposure
at doses of ≥0.3g. In contrast, at 24 h neither fipronil nor imidacloprid reached 90% reduction,
even at the highest doses tested (0.5g for fipronil and 5.0g for imidacloprid). Selamectin was
significantly (P ≤ 0.05) more potent than imidacloprid and fipronil at levels ≥0.03g. A similar
pattern of activity was observed at both 48 and 72 h, but higher percentages of larvae were killed
for each of the compounds as the incubation time increased. At 72 h selamectin was significantly
(P ≤ 0.05) more potent than imidacloprid at levels of 0.01–0.1g and significantly (P ≤ 0.05)
more potent than fipronil at levels of 0.003–0.01g. Therefore, selamectin was more potent than
either fipronil or imidacloprid in killing flea larvae in this in vitro assay system.
© 2003 Published by Elsevier B.V.
Keywords: Selamectin; Fipronil; Imidacloprid; Larval fleas; Ctenocephalides felis; In vitro assay
∗ Corresponding author. Tel.: +1-860-441-6454; fax: +1-860-715-9031.
E-mail address: tom l
mctier@groton.pfizer.com (T.L. McTier).
0304-4017/$ – see front matter © 2003 Published by Elsevier B.V.
doi:10.1016/S0304-4017(03)00163-8
46 T.L. McTier et al. / Veterinary Parasitology 116 (2003) 45–50
1. Introduction
The activity of selamectin (Revolution®/Stronghold®, Pfizer Animal Health), a monosaccharide
avermectin endectocide, against adult cat fleas, Ctenocephalides felis felis, (hereafter
referred to as C. felis) has been well documented (McTier et al., 2000a). The ability
of selamectin to control environmental flea infestation has been demonstrated in several
different types of study designs (Shanks et al., 2000; Dryden et al., 2001a). The potent activity
of selamectin in debris (dander, flea feces, hair) from selamectin-treated dogs against
normal flea eggs and larvae has been demonstrated in vitro (McTier et al., 2000b). In addition,
previous work has shown that selamectin has potent ovicidal activity in vivo (McTier
et al., 2000b; Dryden et al., 2001b). The objective of the present study was to compare the
activity of selamectin, fipronil, and imidacloprid against larval fleas (C. felis) using an in
vitro potency assay.
2. Materials and methods
Selamectin, fipronil and imidacloprid were made up as stock solutions consisting of
500g of active compound per milliliter in acetone. From 8 to 10 different test concentrations
of each compound were formulated from these stock solutions by dilution with
acetone. One hundred microliters of each test solution were added to glass vials (1.5 by
3 cm) to which had been previously added 20 mg of sand and 10 mg of flea feces. The vials
were then ball milled to allow the acetone to evaporate. Selamectin and fipronil were tested
at 0.001, 0.003, 0.005, 0.01, 0.03, 0.05, 0.11, 0.3, and 0.5g of active compounds per tube.
Imidacloprid was tested at 0.01, 0.03, 0.05, 0.1, 0.3, 0.5, 1.0, 3.0, and 5.0g of active
compound per tube. Approximately 30 first instar C. felis larvae were then added to each
vial, the vials were closed with a porous plug and incubated under appropriate conditions
to maintain the flea larvae. The number of live flea larvae remaining in each vial was determined
once daily over a 72 h period following incubation. Four separate replicates were
conducted using the same protocol, and results were combined.
Percentage of dead flea larvae in each tube was transformed using an arcsine square root
transformation of the proportion. Data were analyzed using a mixed model for repeated
measures. Fixed effects included compound, dose, time and the interaction of those effects.
Random effects included residual and replicate. Contrasts among least squares means of
transformed percent death data were used to assess differences among treatment combinations
at each time point. The 5% level of significance (P ≤ 0.05) was used to assess
statistical differences. Efficacy was calculated as a mean percentage of flea larvae killed
from four separate replicates combined.
3. Results
For selamectin, reductions of ≥93.5% were achieved at 24 h after exposure at doses of
≥0.3g per tube (Fig. 1). At 24 h neither fipronil nor imidacloprid reached 90% reduction,
even at the highest doses tested (0.5 g for fipronil and 5.0g for imidacloprid). Selamectin
was significantly (P ≤ 0.05) more potent than imidacloprid and fipronil at levels ≥0.03g.
T.L. McTier et al. / Veterinary Parasitology 116 (2003) 45–50 47
Fig. 1. Activity of selamectin, imidacloprid, and fipronil in killing flea larvae in vitro at 24 h after exposure.
At 48 h selamectin reached the 90% reduction level at a dose of 0.03 g, and a 100%
reduction was achieved at ≥0.1g (Fig. 2). In contrast fipronil did not reach the 90% level
until a 3×higher dose (0.1 g), and imidacloprid did not reach this level until a 100×higher
dose (3.0g). Selamectin was significantly (P ≤ 0.05) more potent than imidacloprid at
all commonly used levels tested (0.01–0.5 g) and significantly (P ≤ 0.05) more potent
than fipronil at levels of 0.003–0.01 g.
Fig. 2. Activity of selamectin, imidacloprid, and fipronil in killing flea larvae in vitro at 48 h after exposure.
48 T.L. McTier et al. / Veterinary Parasitology 116 (2003) 45–50
Fig. 3. Activity of selamectin, imidacloprid, and fipronil in killing flea larvae in vitro at 72 h after exposure.
At 72 h, a pattern similar to that at 48 h was observed. Selamectin killed 98.5% of the
larvae at the 0.03 g level and killed 100% of the larvae at doses of ≥0.05g (Fig. 3).
Reductions of 94 and 100% were observed for fipronil at 0.05 g and≥0.3g, respectively.
Imidacloprid was obviously less potent, with a 98.5% reduction at 0.3 g and a 100%
reduction at ≥0.5g, excluding the 1 g dose. Selamectin was significantly (P ≤ 0.05)
more potent than imidacloprid at levels of 0.01–0.1 g and significantly (P ≤ 0.05) more
potent than fipronil at levels of 0.003–0.01 g.
4. Discussion
Selamectin demonstrated potent activity against flea larvae in this in vitro assay system.
Overall selamectin was more potent than either fipronil or imidacloprid. In this assay, flea
larvae received not only direct contact exposure, but also had the opportunity for systemic
exposure via oral ingestion of the flea feces. Such a design mimics the potential exposure
routes for flea larvae in home environments where pets have been treated with topical
parasiticides. Selamectin’s activity against adult fleas is known to have both systemic and
contact components, with the primary activity probably systemic through ingestion of blood
containing selamectin (Bishop et al., 2000; Melhorn et al., 2001). Imidacloprid’s activity
against adult fleas seems to be primarily through contact, but interestingly fipronil’s activity
seems to be primarily systemic (Melhorn et al., 2001). Flea feces from fleas that have fed on
selamectin-treated animals contain detectable levels of selamectin (Pfizer, data on file), and
debris (flea feces, dander, hair) from selamectin-treated dogs is known to be both ovicidal
and larvicidal (McTier et al., 2000b). For larval fleas in the environment, ingestion of flea
T.L. McTier et al. / Veterinary Parasitology 116 (2003) 45–50 49
feces containing compounds systemically active against larval fleas may represent a more
important route of exposure, as larvae are attracted to adult flea feces as the preferred, even
essential, source of nutrition (Rust and Dryden, 1997). In addition, more eggs, debris and flea
feces tend to fall in areas where the host animal spends the most time, and most flea larvae
remain within the vicinity of where the eggs fall into the environment (Rust and Dryden,
1997). Thus, flea feces containing selamectin would automatically be targeted to areas most
likely to have higher egg and larval flea populations. Therefore, in the environment, flea
larvae would be more likely killed by ingesting feces, containing a drug like selamectin,
to which they are attracted than through incidental contact exposure of larvae to hair and
debris, from a pet treated with a drug like imidacloprid. Several studies have shown that
imidacloprid kills adult fleas faster than selamectin on dogs (Everett et al., 2000; Melhorn,
2000; Melhorn et al., 2001). However, speed of kill appears less critical in actual use
situations, as studies in both dogs and cats have shown that selamectin is as effective as
imidacloprid and fipronil in controlling environmental flea infestation, as assessed by animal
flea counts as well as light traps to capture adult fleas in the environment (Ritzhaupt et al.,
2000a,b; Dryden et al., 2001a). It is likely that a substantial component of selamectin’s
ability to control environmental infestation is due to activity against flea eggs and larvae.
Although selamectin was more potent than either imidacloprid or fipronil in this in vitro
system, care should be taken in extrapolating these results to a product use situation where
dose levels of products to which larval flea populations may actually be exposed are unknown.
However, it is noteworthy that the actual minimum dose actually applied to the
animal as per label instructions is lower for selamectin (6 mg/kg) than for either imidacloprid
(10 mg/kg) or fipronil (7.5 mg/kg).
5. Conclusions
Selamectin was more potent in killing flea larvae in this in vitro assay system than either
fipronil or imidacloprid, achieving higher reductions more quickly at lower doses.
Acknowledgements
This study was funded by Pfizer Inc., New York, NY.
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