236

RESEARCH NOTE
DETECTION OF RESISTANCE TO FIPRONIL IN BROWN DOG TICKS
(Rhipicephalus sanguineus sensu lato) FROM HOUSEHOLD DOGS IN
LAS PIÑAS CITY, PHILIPPINES THROUGH LARVAL PACKET TEST

Francisella P. Katalbas, DVM, Remil L. Galay, DVM, DVSc*, and

Billy P. Divina, DVM, MSc

Department of Veterinary Paraclinical Sciences, College of Veterinary Medicine,

University of the Philippines Los Baños, Laguna 4031, Philippines

ABSTRACT
Tick infestation is among the most common parasitic problems affecting
dogs. Since the 1990s, fipronil has been used in the Philippines for the control of
tick and flea infestations in dogs. Resistance to fipronil has been reported in other
countries but not in the Philippines. This study was conducted to detect fipronil
resistance in the brown dog tick Rhipicephalus sanguineus sensu lato from
household dogs in Las Piñas City, Metro Manila, Philippines. Nearly engorged
female R. sanguineus s.l. were collected manually using a specialized tick
remover. These were allowed to lay eggs in the laboratory where the larvae that
hatched were used for the larval packet test (LPT) with three different doses of
fipronil: recommended dose (0.25%), half of the recommended dose (0.125%), and
double the recommended dose (0.50%). Additionally, three fipronil commercial
brands were subjected to LPT using the commercial dose to compare their effect
on larval mortality. The larval mortality in all fipronil doses and commercial
brands ranged from 91.6% to 100%, suggesting the absence of fipronil resistance.
This indicates that fipronil is still useful in controlling tick infestation in dogs in
Las Piñas City however, more studies covering other areas should be done to
determine the status of resistance in the country.

Key words: acaricide resistance, dog, fipronil, larval packet test, Rhipicephalus sanguineus
Philipp. J. Vet. Med., 57(2): 236-242, 2020

INTRODUCTION
Pet ownership in the Philippines has
been steadily increasing over recent years. Many
Filipinos own at least one pet dog per household
(Santiago, 2010). It has also been estimated that
there is one pet dog for every eight people
(Olivarez, 2014). Increasing dog populations
however, can lead to increased disease incidence
and transmission. Among the commonly
encountered problems by dog owners in the
Philippines is the presence of ticks. Tick
infestation makes dogs aesthetically unpleasing
and affect their overall health and well-being
through direct effects and transmission of
pathogens.
Rhipicephalus sanguineus sensu lato is
the most widespread tick in the world and a well-
recognized vector of many pathogens affecting
dogs and occasionally, humans (Dantas-Torres,
2010). The common pathogens transmitted by R.
sanguineus s.l. are Babesia spp., Anaplasma
platys, Ehrlichia canis, and Hepatozoon canis,
some of which are potentially zoonotic
(Dantas-Torres and Otranto, 2015). In the
Philippines, several studies already showed the
prevalence and diversity of tick-borne pathogens
in dogs, with concurrent infection being common,
especially in Metro Manila (Galay et al., 2018).
Chemical acaricides have been widely used
for controlling tick populations (Nandi et al.,
2015). There are several acaricides used in
controlling dog ticks including fipronil
(phenylpyrazole), amitraz (amidine), carbaryl
(carbamate), and permethrin (synthetic
pyrethroid) (Dantas-Torres, 2008).
*FOR CORRESPONDENCE:
(e-mail: rlgalay@up.edu.ph)
 RESISTANCE TO FIPRONIL IN Rhipicephalus
KATALBAS et al
Newer acaricides that have been developed
are afoxolaner and fluralaner, from the class
isoxazoline, in the form of a chewable tablet. The
misuse of acaricides such as incorrect preparation
and application, can lead to the failure of
eliminating tick burdens from an animal (FAO,
2004). However, long term use and misuse of
acaricides can lead to the development of
resistance in ixodid ticks (Dantas-Torres, 2008).
One of the most common chemical
acaricides currently utilized in controlling dog
ticks and fleas is fipronil. Fipronil is a
phenylpyrazole compound used in controlling dog
ticks (Jacobs et al., 2016). It blocks the
gamma-amino-butyric acid (GABA) gated chloride
channels leading to hyperexcitation of the central
nervous system of the tick (APVMA, n.d.). In the
Philippines, it has been in use since the 1990s. It
is available as spray and spot-on preparations.
In August 2010, fipronil patent protection
ended and generic formulations have become
available to consumers at a lower price (Eiden et
al., 2015a). Recent studies on tolerance of dog
ticks to fipronil have been reported in other
countries. This tolerance was attributed to the
emergence of generic formulations (Eiden et al.,
2017). Increased availability and low cost of
generic formulations of fipronil can lead to
increased exposure of R. sanguineus s.l. to
fipronil, inevitably causing failure of the product
due tolerance and resistance through developing
the ability to tolerate doses of toxicants lethal to
the majority of the normal population (WHO,
2012). Thus, there is a need to determine whether
dog ticks in the Philippines are already resistant
to fipronil.
This study aimed to determine the presence
of resistance against fipronil in R. sanguineus s.l.
ticks infesting dogs from households in Las Piñas
City, a residential, commercial, and industrial
suburb in Metro Manila, Philippines, with an
increasing human population and a high
population of dogs. A recent study showed the
high detection of tick-borne pathogens in dogs in
Las Piñas City (Galay et al., 2018). Knowledge of
resistance status to fipronil will be helpful in
deciding the appropriate tick control method,
which is essential in preventing the spread of
tick-borne pathogens.
MATERIALS AND METHODS
Study Population
Collection of ticks was done in nine
barangays in Las Piñas City, Metro Manila,
Philippines from February 2018 to January 2019.
At least five nearly engorged ticks were collected
sanguineus 237
per dog. A total of 85 nearly engorged female
ticks collected from 17 dogs were used to produce
larvae that were subjected to acaricide resistance
test. Information on dogs including the name,
age, sex, breed, living environment, history of
tick infestation, chemical acaricides used, method
of acaricide application, frequency of acaricide
application, and date of last acaricide application
was obtained through personal interview of the
dog owners.
Tick Collection and Identification
The protocol for obtaining the ticks from
dogs was approved by the Institutional Animal
Care and Use Committee of the College of
Veterinary Medicine, University of the
Philippines Los Baños (protocol number
2018-0004). Female ticks were located
throughout the body of the dog including the
common infestation sites: the head, base of the
ear, back, inguinal region, axilla, and interdigital
spaces of the dog (Dantas-Torres, 2010). Ticks
were manually detached using a specialized tick
remover (O’tom/Tick Twister® H3D, Lavancia,
France). Collected ticks were placed in labeled
small glass vials with slightly moistened cotton
ball to keep the humidity requirement and
protect ticks from desiccation (FAO, 2004).
Immediately upon arrival at the laboratory,
engorged female ticks were washed with diluted
phosphate-buffered saline (PBS) to remove dirt.
Ticks were examined under a stereomicroscope to
confirm their identity based on morphological
characteristics described by Barker and Walker
(2014), and to check if mouthparts were still
intact. Ticks that have damaged mouthparts or
have already started laying eggs were discarded.
Preparation of tick larvae
The female ticks were placed in individual
wells of a 24-well cell culture plate. The plate
was then stored in a sealable plastic ware with a
damp paper towel at the bottom and was kept at
room temperature for three weeks or until egg
laying has ended (FAO, 2004). Once egg laying
has ended, eggs were carefully transferred to the
prepared sterile test tubes, wherein eggs of ticks
collected from the same dog were pooled. The test
tubes were kept at room temperature until all
the eggs have hatched. The larvae were subjected
to the larval packet test (LPT) within 14-21 days
from hatching.
Bioassay for fipronil resistance and brand
susceptibility
For economic reasons, LPT for acaricide
238 KATALBAS
RESISTANCE TO FIPRONIL IN et. al
Rhipicephalus sanguineus
resistance was adapted from the studies of Eiden
et al. (2015a) and Vudriko et al. (2016), both of
which were modified from the standard LPT
procedures set by FAO (2004).
For resistance test, three concentrations
of a commercial grade, spot-on preparation of
fipronil (Fiproxil®, United Pharma Ltd.,
Budapest, Hungary) were prepared with absolute
ethanol: commercial spray preparation or the
recommended dose (0.25%), half of the
recommended dose (0.125%), and double the
recommended dose (0.50%). For each dose, 0.8 ml
was applied to a piece of 8.5 cm x 8.5 cm filter
paper (Whatman® no. 1, GE Healthcare’s Life
Sciences, Singapore, Singapore) and was allowed
to dry under a fume hood for three hours. For
negative control, 0.8 ml of absolute ethanol was
applied to a separate filter paper. Each filter
paper was folded into packets. Approximately 50
larvae were placed in each packet. Each packet
was sealed with double-adhesive tapes and
secured with metal binder clips and then placed
in a sealable plastic ware that was lined with
damp paper towel and aluminum foil. Two
replicates were done per concentration. The
packets were kept at room temperature for 24
hours after which they were opened to determine
larval mortality.
For determination of variation in larval
mortality among commercial fipronil spray
preparations, three brands available in the local
market with 0.25% concentration, Brand X (Flea
+ Tick Defense®, Vetri-Science Laboratories,
Washington, United States of America), Brand Y
(Frontline®, Merial SAS, Lyon, France), and
Brand Z (Ticks Away®, Tashuo Nippon Pharma
Co., Osaka, Japan), were used. For each of the
commercial spray preparations, 0.8 ml was
applied directly unto the filter paper and LPT
was performed as described above. LPT using
different concentrations and different brands
were done simultaneously.
Identification of dead tick larvae was based
on the inability of tick larvae to walk after being
gently nudged using a soft paintbrush and by
gently breathing directly onto the larvae to
stimulate movement (FAO, 2004). Only larvae
capable of walking were considered to be alive.
Observation under the stereomicroscope was
done to assess the larvae more thoroughly. After
checking for the mortality, all packets with the
larvae were autoclaved to ensure that all larvae
were killed before disposal.
Data Analysis
Post-LPT mortality in each packet was
calculated by dividing the number of dead larvae
by the total number of larvae expressed in
percentage, and then the average percent
mortalities per concentration of fipronil and
fipronil brand were calculated. Interpretation of
resistance was based on the recommendation of
FAO (2004) wherein larval pools with at least
80% mortality in recommended dose were
considered susceptible while those that had less
than 80% mortality were considered resistant.
Student’s t-test was performed to
determine whether there are significant
differences (α = 0.05) between the control and the
different doses and brands of fipronil on tick
mortalities and in comparing larval pools of ticks
that came from dogs with a history of fipronil
treatment and those without history. Analysis of
Variance (ANOVA) was also done using the
online software OpenEpi® to test for the
significant differences (α = 0.05) in larval
mortalities after exposure to different brands.
RESULTS AND DISCUSSION
Effective tick control is essential in
controlling tick-borne infections in dogs. There
are some anecdotal claims by dog owners that
some commercially available chemical acaricides
are already ineffective in controlling tick
infestation. Thus, the present study was
conducted to determine whether there is
resistance already to fipronil, a commonly used
acaricide in dogs from selected households in Las
Piñas City, Metro Manila, Philippines, through
LPT, as well as to evaluate the susceptibility to
three different commercial brands of fipronil.
The dogs from which engorged female
ticks were collected came from nine barangays in
Las Piñas City, comprised of eight different
breeds, and majority (13/17) were kept indoors
(Table 1). The majority (14/17) of the owners
employed manual removal of ticks as an adjunct
to or as sole means of tick control. Out of the 17
dogs sampled, seven owners reported to having
used fipronil spot-on as tick control. Through the
interview, there were some owners who
mentioned that fipronil seemed ineffective in
controlling tick infestation. Aside from fipronil,
carbaryl powder and afoxolaner tablet were used
individually by two different dog owners while
eight have not used any chemical acaricides.
Most of the dog owners who administered
chemical acaricide reported using them only
when they find ticks in their dogs.
LPT using three different concentrations
of fipronil showed 100% mortality in the majority
of the larval pools tested except for three
 RESISTANCE TO FIPRONIL IN Rhipicephalus
KATALBAS et al sanguineus 239

Table 1. Summary of selected attributes of 17 household dogs in Las Piñas City from where
female Rhipicephalus sanguineus sensu lato ticks were collected.
Attributes Number of Dogs
Pomeranian 1
Shih Tzu 2
Beagle 2
Pitbull 1
Breed
Labrador Retriever 2
Belgian Malinois 1
Mixed Breed 8
Total 17
Indoor 13
Type of living
Outdoor 4
environment
Total 17
Carbaryl 1
Fipronil 7
Acaricides used Afoxolaner 1
None 8
Total 17
Powder 1
Spot-on 7
Preparation of
Tablet 1
acaricide used
None 8
Total 17
Every week 1
Frequency of Every 3 months 1
acaricide When ticks are present 7
application None 8
Total 17
Manual removal 14
Other means of tick
None 3
control
Total 17

Table 2. Average percent (%) mortality of Rhipicephalus sanguineus sensu lato tick larvae that were
subjected to larval packet test (LPT) using three different concentrations or doses of fipronil.
Fipronil Dose and Average % Mortality
Larval
Control Half dose Recommended Double dose
Pool
(diluent) (0.125%) dose (0.25%) (0.50%)
1 0 91.6 95.3 97.6
2 3.7 100 100 100
3 4.9 100 100 100
4 0 100 100 100
5 0 98.1 99.1 99
6 3 100 100 100
7 0 100 100 100
8 3.4 98.2 100 100
9 0 100 100 100
10 0 100 100 100
11 0 100 100 100
12 0 100 100 100
13 0 100 100 100
14 1.7 100 100 100
15 0 100 100 100
16 0 100 100 100
17 0 100 100 100
*All treated groups are significantly different (α = 0.05) from the control based on
Student’s t-test.
(Table 2). Larval pools 1 and 5 had mortality before LPT. Meanwhile, all larval pools showed
ranging from 91.6% to 99% in three different dos- more than 80% mortality for each fipronil
es of fipronil, whereas larval pool 8 had 98.2% concentration tested suggesting the absence of
mortality in the half dose of fipronil. All larval resistance based on FAO (2004) standard. Only
pools showed percent mortality below 10% when two larval pools had a mortality of less than
exposed to the negative control (absolute ethanol 100% in the recommended dose, albeit still above
only) indicative of the good condition of larvae 90%. One of these two pools came from the ticks
240 KATALBAS
RESISTANCE TO FIPRONIL IN et. al
Rhipicephalus sanguineus

Table 3. Average percent (%) mortality of Rhipicephalus sanguineus sensu lato tick larvae that were
subjected to larval packet test (LPT) using the recommended dose (0.25%) of three commercial brands
of fipronil.
Average % Mortality*
Larval Pool
Brand X Brand Y Brand Z
1 100 94.2 99.2
2 100 99.0 100
3 100 98.0 100
4 100 100 100
5 99.0 98.1 95.7
6 100 98.3 100
7 100 100 100
8 98.0 100 100
9 100 98.1 100
10 100 100 100
11 100 100 100
12 100 100 100
13 100 100 100
14 100 99.2 100
15 100 100 100
16 100 100 100
17 100 100 100
*All brands are significantly different (α = 0.05) from the control (same as in Table 2)
based on Student’s t-test, but ANOVA showed no significant differences (α>0.05) among
the three brands.

collected from a dog that has been treated with
fipronil. Student’s t-test indicated that the larval
mortalities in all fipronil-treated groups were
significantly higher compared to the control.
Regarding LPT using different commercial
brands of fipronil spray, the majority of the larval
pools showed a 100% average mortality (Table 3).
Two larval pools exposed to Brand X,
seven larval pools exposed to Brand Y, and one
larval pool exposed to Brand Z had mean
mortalities ranging from 94.2 to 99.2%. At 80%
mortality as an indication for fipronil
susceptibility, all the commercial brands of
fipronil tested are still effective for tick control.
The percent mortalities in all commercial
preparations of fipronil were significantly
different compared to the control based on
Student’s t-test. While the LPT results in the
three fipronil brands seemed to suggest a
difference in susceptibility, ANOVA illustrated
that there was no statistical significance
(Table 3).
Based on Student’s t-test, there were no
significant differences in the mortalities of larval
pools from dogs previously treated with fipronil
and not previously treated with fipronil (Table 4).
Larval pools from dogs whose owners have
suspected that fipronil was no longer effective in
tick control all showed 100% average mortality,
contradicting their claim. It is important to note
that most of the owners admitted that they were
not following the recommended monthly
application of fipronil and only applied when they
saw ticks in their dogs. Since all of the
commercial preparations of fipronil are labeled
for a once-monthly application, it should strictly
be followed to achieve desired efficacy against
Tolerance of the brown dog ticks to fipronil was
first described in a study in the
United States wherein low resistance ratio was
observed in some strains (Eiden et al., 2015a).
More recently, confirmed resistance to fipronil in
R. sanguineus s.l. has been reported in a brown
dog tick population in Brazil (Becker et al., 2019).
While resistance was not detected in this study,
dog owners should be educated on the proper
application of fipronil to prevent the emergence
of resistance in the future.
In conclusion, resistance to fipronil was
not detected in R. sanguineus s.l. larvae from
engorged female ticks collected from household
dogs in Las Piñas City through LPT. It was also
found that the larvae are susceptible to three
different commercial preparations, with no
statistically significant difference in
susceptibility. These results suggest that fipronil
can still be used for the control of tick infestation
in dogs following the recommended application
method and frequency. Nevertheless, the results
of this study do not imply the absence of
resistance to fipronil in dog ticks from other
areas hence, further studies are needed. To the
authors’ knowledge, this is the first report
investigating the occurrence of fipronil resistance
in dog ticks in the Philippines.
 RESISTANCE TO FIPRONIL IN Rhipicephalus
KATALBAS et al sanguineus 241

Table 4. Mean (±S.D.) percent (%) mortality of larvae of Rhipicephalus sanguineus sensu lato
femaleticks from dogs with and without a previous history with fipronil treatment. n = number of
larval pools
Fipronil Dose and Brand and Mean % Mortality*
History of Dose Brands (0.25%)
fipronil
use Double
Half dose Recommended Brand Brand
Dose Brand Z
(0.125%) Dose (0.25%) X Y
(0.50%)
Has used
99.7 99.9 99.9 99.5
fipronil 99.9 (0.36) 99.4 (1.62)
(0.71) (0.38) (0.38) (0.76)
(n=7)
Has not
used 99.8 99.8 98.9
99 (2.67) 99.5 (1.49) 99.9 (0.43)
fipronil (0.77) (0.62) (1.86)
(n=10)
*In a column, Student’s t-test at α = 0.05 did not reveal a significant
difference between larval pools produced by female ticks from dogs
with and without a history of fipronil treatment.

ACKNOWLEDGMENT animals and humans. New Zealand:

This study was funded by the Balik PhD
grant of the University of the Philippines. The
authors are grateful to Dr. August Michael
Basangan of the City Veterinarian’s Office of Las
Piñas City, Dr. Kristina Andrea Sandalo, and Dr.
John Michael G. Bernardo for assistance in the
conduct of experiments, and to Dr. Marianne
Leila S. Flores and Dr. Rey B. Oronan for their
technical assistance.
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