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First report of fluazuron resistance in rhipicephalus microplus: a field tick population resistant to six classes of acaricides
Contents lists available at
First report of ﬂuazuron resistance in Rhipicephalus microplus:
A ﬁeld tick population resistant to six classes of acaricides
José Reck , Guilherme Marcondes Klafke , Anelise Webster,
Bruno Dall'Agnol, Ramon Scheffer, Ugo Araújo Souza,
Vivian Bamberg Corassini, Rafael Vargas, Julsan Silveira dos Santos,
João Ricardo de Souza Martins
Instituto de Pesquisas Veterinárias Desidério Finamor (IPVDF), Fundac¸ão Estadual de Pesquisa Agropecuária (FEPAGRO), Eldorado do
The control of the cattle tick Rhipicephalus microplus is based mainly on the use of chem-
Received 11 September 2013
ical acaricides, which has contributed to the emerging problem of selection of resistant
Received in revised form 7 January 2014
tick populations. Currently, there are six main classes of acaricides commercially avail-
Accepted 19 January 2014
able in Brazil to control cattle ticks, with ﬂuazuron, a tick growth regulator with acaricidal
properties, being the only active ingredient with no previous reports of resistance. Ticks
(designated the Jaguar strain) were collected in a beef cattle ranch located at Rio Grande do
Sul state, Southern Brazil, after a complaint of ﬂuazuron treatment failure. To characterise
the resistance of this strain against acaricides, larval tests were performed and showed that
the Jaguar strain was resistant to all of the drugs tested: cypermethrin (resistance ratio,
RR = 31.242), chlorpyriphos (RR = 103.926), ﬁpronil (RR = 4.441), amitraz (RR = 11.907) and
ivermectin (3.081). A ﬁeld trial was conducted to evaluate the efﬁcacy of ﬂuazuron treat-
ment in heifers that had been experimentally infested with the Jaguar or a susceptible
strain. Between 14 and 28 days after treatment, the average efﬁcacy in cattle experimen-
tally infested with the susceptible strain was 96%, while for the Jaguar strain the efﬁcacy
was zero. Additionally, the Jaguar strain response to ﬂuazuron was evaluated in vitro using
a modiﬁed adult immersion test (AIT) and the artiﬁcial feeding assay (AFA). With the AIT,
50 ppm of ﬂuazuron inhibited 99% of larvae hatching in the susceptible strain (POA) and
less than 50% in the Jaguar strain. Results of the AFA showed a larval hatching rate of 67% at
2.5 ppm of ﬂuazuron with the Jaguar strain; conversely, only 3% of larvae of the susceptible
strain hatched at the same ﬂuazuron concentration. The results showed here demonstrated
the ﬁrst case of ﬂuazuron resistance in R. microplus and the ﬁrst tick population resistant
to six classes of acaricides in Brazil.
2014 Elsevier B.V. All rights reserved.
∗ Corresponding author at: Laboratório de Parasitologia, Instituto de Pesquisas Veterinárias Desidério Finamor (IPVDF), Fundac¸ão Estadual de Pesquisa
Agropecuária (FEPAGRO), Estrada do Conde, 6000, Eldorado do Sul 92990-000, RS, Brazil. Tel.: +55 51 3481 3711; fax: +55 51 3481 3711.
E-mail addresses: (G.M. Klafke).
These authors contributed equally for the conduction of this study.
0304-4017/$ – see front matter 2014 Elsevier B.V. All rights reserved.
J. Reck et al. / Veterinary Parasitology 201 (2014) 128–136
tick-resistant populations, particularly in Southern Brazil
The cattle tick Rhipicephalus microplus is a major cause of
to describe the ﬁrst report of ﬂuazuron resistance in R.
concern for cattle breeding in the tropical and subtropical
microplus, and also report the ﬁrst tick strain that was
world, particularly in Latin America. Its parasitism reduces
multi-resistant to six classes of acaricides.
weight gain, causes anaemia, increases the risk for myia-
sis occurrence, and can also result in the transmission of
2. Materials and methods
Babesia bovis, B. bigemina and Anaplasma marginale, which
are the causative agents of cattle tick fever (
2.1. Tick strains
Currently, tick control
is mainly, if not exclusively, based on the use of chemical
2.1.1. Jaguar strain
Ticks were collected from beef cattle at a ranch located
The control of cattle ticks with chemical compounds
in the municipality of Eldorado do Sul, RS, Southern Brazil.
started at the end of the nineteenth century with the use
This farm only breeds Bos taurus taurus cattle, and apart
of arsenic The use of this compound was
from raising beef cattle, it breeds bulls for sale. Addition-
followed by the use of organochlorines (OC) in the 1940s,
ally, it has an intense animal trade activity with other
and a few years later, resistance to this class was reported.
ranches, exchanging cattle with several other properties
Subsequently, these were replaced by organophosphates in
in RS and others states of Brazil. Since 2010, there has been
the 1950s (According to the Brazilian Com-
a suspicion of ﬂuazuron treatment failure as reported by
pendia of Veterinary Products (there are
property staff. In the past years, cattle have been heavily
currently six main classes of chemical acaricides marketed
infested with ticks at this location, particularly during the
for tick control in Brazil: (i) organophosphates (OP), (ii) for-
late summer and mid-autumn (February to May). Tick con-
mamidines (amitraz), (iii) synthetic pyrethroids (SP), (iv)
trol had been based on ﬂuazuron treatments (Acatak®
macrocyclic lactones (ML), (v) phenylpyrazoles (ﬁpronil),
Pour On, Novartis Saúde Animal, Barueri, SP, Brazil),
and (vi) benzoylphenyl ureas (ﬂuazuron). The large scale
pour-on application and dipping vats with a mixture of
and frequent use of acaricides aiming to control ticks on
chlorpyrifos and cypermethrin (Colosso® Pour On and
cattle has favoured the selection of tick populations that are
Colosso® Pulverizac¸ão, Ouroﬁno Saúde Animal, Cravinhos,
resistant to several active ingredients (AI) of commercial
SP, Brazil), and the periodic use of long-acting formula-
acaricides. In recent years, there have been an increasing
tions of avermectins. This property had a history of a high
number of products in the Brazilian market that are mix-
level of tick resistance to cypermethrin, deltamethrin, ami-
tures of two or more AIs
traz, mixture of cypermethrin + chlorpyriphos, and mixture
Reports of resistance against the main classes of aca-
of cypermethrin + ethion, according to data from previous
ricides, particularly for OP, SP, and amitraz, have been
adult immersion tests (AIT) conducted at IPVDF (data not
published in the regions in which the cattle tick is found
shown). On February 11th, 2011, ticks from this ranch were
sampled to establish a laboratory colony. At that time, the
As a result, the use of ML, ﬁpronil,
frequency of treatment with ﬂuazuron was shorter than
ﬂuazuron, and mixtures of SP and OP has become common.
every six weeks, and the frequency of treatments using
The ﬁrst report of OP resistance was registered in Australia
mixtures of organophosphate and pyrethroid was less than
(and eight years later in Brazil
every three weeks. Even with these practices to attempt
(Amitraz started to be used in 1975 in
control, the majority of cattle was observed to be highly
Australia and in 1977 in Brazil, and acaricide resistance
infested with all stages of R. microplus. Some bulls had more
to this chemical class was reported in Australia by 1981
than 700 adult ticks. Ticks referred to as "Jaguar R" were
(Twelve years later, amitraz resistance
obtained by the selection of ticks which survived the ﬁrst
was documented in Brazil SPs were
AIT bioassays, as described below.
introduced in the early 1980s, and resistance was simul-
taneously detected by 1989 in both Australia and in the
2.1.2. São Gabriel (SG) strain
state of Rio Grande do Sul (RS), Brazil
Ticks were obtained from cattle at FEPAGRO Experimen-
MLs became commercially available
tal Station of São Gabriel, municipality of São Gabriel, RS,
in 1981 and resistance in Brazil was ﬁrst reported in 2001,
Brazil (30◦20 S, 54◦15 W). This experimental station has
once again in the state of RS (
a closed herd, i.e. it was maintained without the introduc-
Fipronil has been on the market since 1996, and within
tion of cattle from other farms for more than 10 years. The
a decade, between 2004 and 2006, the ﬁrst report of in
tick population from this herd has a history of resistance
vitro resistance was published in RS, Brazil
against synthetic pyrethroids, amitraz and macrocyclic lac-
One year later, ﬁpronil resistance was conﬁrmed in
tones however, it was never
a stall test in Uruguay Fluazuron is a
exposed to ﬂuazuron, so it was considered a susceptible
benzoylphenylurea derivative that regulates tick growth
ﬁeld strain for this study.
by inhibiting chitin incorporation into the tick's cuticle,
acts systemically, and has acaricidal properties; this was
2.1.3. Porto Alegre (POA) strain
released in the market in 1994, and to date had been
These ticks were collected from a ranch along the
considered the only AI without any evidence of resistance,
border of Brazil and Uruguay in 1992. It has been kept at
despite the remarkable increase in its use to control cattle
Porto Alegre municipality, RS, Brazil, in the facilities of
J. Reck et al. / Veterinary Parasitology 201 (2014) 128–136
Universidade Federal do Rio Grande do Sul (UFRGS). The
0.00195, 0.0039, 0.0078, 0.0156, 0.0312, 0.0625, 0.125, 0.25
POA strain has been widely used as a susceptible reference
tick strain since its isolation more than twenty years ago,
and has been maintained without exposure to acaricides.
(85 mm × 75 mm – Whatman No. 1, Whatman Inc.,
All tick strains/colonies were maintained in stall con-
Maldstone, England), or six nylon fabrics in the case of
ditions at the Isolation Unit of Instituto de Pesquisas
amitraz (85 mm × 75 mm – Type 2320, Cerex Advanced
Veterinárias Desidério Finamor (IPVDF), Eldorado do Sul,
Fabrics, Pensacola, FL), were prepared (three for each
RS, Brazil, without exposure to other contaminant tick
strain). Filter papers were impregnated with 0.67 ml each
populations or acaricides as previously described
of the solutions using a micropipette. The material was left
to dry for 2 h inside a fume hood to allow trichloroethylene
evaporation. After drying, the ﬁlter papers were folded
2.2. Larval bioassay
in the middle and sealed on the sides with a metal clip
to form the packets. Approximately 100 larvae were
Determination of the magnitude of resistance to differ-
transferred to each packet using a No. 2 paintbrush. The
ent classes of acaricides was accomplished using a larval
packets were sealed with a third clip and incubated at
bioassay with the Jaguar and POA strains. For cypermethrin,
27–28 ◦C and 80–90% relative humidity. The control group
chlorpyriphos and ﬁpronil, the larval packet test (LPT) was
was exposed to ﬁlter paper treated with acaricide-free
performed according to procedures established by
TCE-OO. After 24 h, larvae mortality was determined by
toxicity was evaluated with the larval
counting the total dead and alive individuals. Larvae that
immersion test (LIT) as described by
were paralysed or moving their legs without the capability
The modiﬁed LPT was conducted according to
to walk were considered dead.
amitraz. All of the larval tests were performed
simultaneously with the susceptible POA strain.
2.5. Larval immersion test
The tests were conducted with technical grade cyper-
methrin, chlorpyriphos, ivermectin (Sigma Chemical Co.,
Initially, a solution of 2% Triton X-100 (Sigma Chemical
St. Louis, MO, USA), and ﬁpronil (BASF Chemicals, Paulínia,
Co., St. Louis, MO, USA) was prepared in absolute ethanol
SP, Brazil). The tests with amitraz were done using a com-
(Merck, Darmstadt, Germany) (ETH-TX2%). Technical iver-
mercial formulation at 12.5% (Triatox®, MSD Saúde Animal,
mectin was diluted to 1% in 10 ml of the ETH-TX2% solution
São Paulo, Brazil).
in order to prepare a stock solution. At the time of testing,
100 L of the stock solution was added to 9.9 ml distilled
2.3. Preparation of ticks
water to obtain the following ﬁnal concentrations in the
mixture: 0.01% ivermectin, 1% ethanol, and 0.02% Triton
Engorged females of the POA and Jaguar strains were
X-100. This mixture was serially diluted 10 times at a
collected following normal detachment from the host.
30% rate in a diluent composed of 1% ethanol and 0.02%
Ticks were processed in the laboratory according to FAO
Triton X-100, in order to obtain the ﬁnal immersion solu-
procedures (Brieﬂy, after being washed with
tions with the following concentrations (in % of AI): 0.01,
water and dried with paper towels, the ticks were incu-
0.007, 0.0049, 0.00343, 0.0024, 0.00168, 0.00117, 0.00082,
bated in plastic Petri dishes (90 mm diameter × 22 mm
0.00057, 0.0004 and 0.00028. Diluent without acaricide
high) inside an environmental chamber, in the dark at
was used as a control. Five hundred microlitres of each
temperatures between 27 and 28 ◦C and relative humid-
immersion solution was distributed in three 1.5 ml micro-
ity between 85 and 90% for two weeks to allow oviposition.
centrifuge tubes (Axygen, Union City, CA, USA). Using a No.
Egg masses were thoroughly mixed and then placed in glass
2 paintbrush, approximately 100 larvae were transferred
vials (5 ml) closed with a cotton lid to allow air and humid-
to each tube, which was then closed and shaken vigorously
ity passage. Eggs in the vials were incubated under the same
to ensure sinking of the larvae. After 10 min of immer-
conditions as the adult females to allow the emergence of
sion, the larvae were removed from the tube with a clean
larvae. Larvae used for testing were between 14 and 21 days
paintbrush and allowed to dry on a piece of paper towel,
before being transferred to a packet of ﬁlter paper folded in
the middle and closed on the sides with metal clips. After
2.4. Larval packet test
adding the larvae, the packet was sealed with a third clip
and incubated in an environmental chamber at 27–28 ◦C
Stock solutions of cypermethrin, chlorpyriphos, ﬁpronil
and 85–90% relative humidity in the dark. After 24 h, larvae
and amitraz were prepared in a mixture containing two
mortality was determined by counting the total number of
parts trichloroethylene (Synth, Diadema, Brazil) and one
dead and alive individuals. Larvae that were paralysed or
part commercial olive oil (TCE-OO). These stock solutions
moving only their legs without the capability to walk were
were used to prepare the following impregnation solutions
in TCE-OO (in % of AI): (i) Cypermethrin 0.01, 0.015, 0.02,
0.03, 0.04, 0.08, 0.1, 0.2, 0.3, 0.4, 0.6, 0.8, 1, 2, 3 and 4;
(ii) Chorpyriphos 0.003, 0.006, 0.01, 0.02, 0.03, 0.04, 0.06,
0.3, 0.6, 0.8, 1, 2, 3 and 4; (iii) Fipronil 0.00008, 0.00016,
A total of 20 heifers (Bos taurus taurus, Angus breed),
0.00032, 0.0006, 0.0012, 0.0025, 0.005 and 0.01; (iv) Ami-
aged 18–20 months, were used. Cattle were divided
traz 0.000061, 0.000122, 0.000244, 0.000488, 0.000976,
into four groups (ﬁve animals each) maintained in four
J. Reck et al. / Veterinary Parasitology 201 (2014) 128–136
paddocks (≈1 ha each) separated by electric fences at the
in demineralised sterile water) to give four additional test
IPVDF facilities (30◦0306 S; 51◦1844 W), Eldorado do
concentrations (in ppm of ﬂuazuron): 50, 5, 0.5, and 0.05.
Sul, RS, Brazil. Cattle were allowed to graze freely on nat-
Thirty engorged females of each strain (POA, Jaguar and
ural pasture and had free access to water. The cattle were
Jaguar R) were immersed in 30 mL of each solution for
handled in accordance with local institutional guidelines
1 min. The treated ticks were then dried with a paper
and all procedures were in accordance with international
towel and incubated individually in glass tubes (5 mL) at
guidelines for animal farm experimentation
27 ± 1 ◦C and 80% relative humidity for six weeks. Follow-
At days −21, −14, −7 and −1, all animals were infested
ing egg hatching, the proportion of larvae was estimated
with approximately 20,000 larvae (14–21 days old) of
by visual inspection using a stereomicroscope performed
Jaguar (resistant) or SG (susceptible) strains, as described
by the same trained operator.
below. Four homogeneous groups were formed by a ran-
domised block design (higher to lower tick count) based
2.8. Artiﬁcial feeding assay (AFA)
on the average number of adult ticks on the left side of
each animal counted at days
As ﬂuazuron is a systemic acaricide and ticks become
−2, −1 and zero. The four experimental groups were:
exposed when they feed on blood, experiments of arti-
(1) Jaguar-infested heifers (control); (2) Jaguar-infested
ﬁcial feeding were conducted as an alternative in vitro
heifers treated with ﬂuazuron; (3) SG-infested heifers (con-
assay to characterise the toxicity of ﬂuazuron between
trol); (4) SG-infested heifers treated with ﬂuazuron.
tick strains. Artiﬁcial feeding was performed as described
On day 0, animals from groups 2 and 4 were treated
by Brieﬂy, partially engorged female
with ﬂuazuron (Acatak®, Novartis Saúde Animal, Barueri,
ticks (between 30 and 80 mg) from the POA and Jaguar
SP, Brazil), according to the manufacturer's instructions, i.e.
strains were manually collected 19 days after larvae were
2.5 mg/kg (5 ml/50 kg). No rain was recorded in the days
used to experimentally infest heifers housed in individ-
after the treatments.
ual pens. Blood for artiﬁcial feeding was obtained from a
Adult tick counts (female ticks >4.5 mm) on the left side
pool of samples collected with sodium citrate from three
of heifers, done according to a previously established pro-
heifers maintained without previous acaricide exposure.
tocol were determined on days
Blood was placed in a microhaematocrit capillary tube
7, 14, 21 and 28. Adult tick counts are reported as the
(without heparin) that was placed at the tick's hypos-
mean ± S.E. of ﬁve animals.
tome. Treatments included: (i) blood, (ii) blood + ﬂuazuron
For analysis of treatment efﬁcacy, the reproductive
solution (ﬁnal concentrations 2.5, 5 and 10 ppm), and (iii)
parameters of collected engorged females were assessed
blood + vehicle (0.02% Triton X-100 in 1% acetone in dem-
according to WAAVP guidelines for evaluation of the efﬁ-
ineralised sterile water). Twenty ticks were used for each
cacy of acaricides Brieﬂy, at least
treatment. Fluazuron solution preparation was performed
10 fully engorged female ticks were collected on days 7,
as described above. Ticks were fed for approximately 24 h.
14, 21 and 28 from each heifer for evaluation of egg mass
The capillary tubes were replaced with new ones when
weight and larvae hatching. Ticks collected from experi-
empty (approximately every 2 h). During artiﬁcial feeding,
mental animals were washed with water, dried in a paper
ticks were maintained in incubators at 27 ± 1 ◦C and 80%
towel, and placed in Petri dishes inside an environmen-
relative humidity. Upon repletion, ticks were weighed and
tal chamber at 27 ± 1 ◦C and 80% relative humidity for two
held individually in glass tubes at 27 ± 1 ◦C and 80% relative
weeks. Thereafter, eggs were removed and transferred to
humidity for six weeks. The proportion of larvae emerg-
glass tubes sealed with cotton plugs and maintained under
ing from hatched eggs was estimated by visual inspection
the same conditions described for females until all the
using a stereomicroscope performed by the same trained
larvae hatched, which occurred approximately 4–6 weeks
later. Larvae were enumerated by visual inspection using a
stereomicroscope performed by the same trained operator.
2.9. Statistical analysis
2.7. Adult immersion test (AIT) with ﬂuazuron
Statistical signiﬁcance of the treatments in the ﬁeld trial
was analysed by the Student's t test. Statistical analyses for
the AIT and AFA were calculated using one-way analysis
procedure to evaluate the relative sensitivity of R. microplus
of variance (ANOVA) followed by Tukey's multiple com-
to ﬂuazuron. The FAO Guidelines for Resistance Manage-
parison test. P values < 0.05 were considered statistically
ment and Integrated Parasite Control in Ruminants (
different. Student's t test and ANOVA were performed using
suggest that this method could be improved and
GraphPad Prism 3.0 (GraphPad Software Inc., San Diego, CA,
adapted for testing resistance. Brieﬂy, technical grade ﬂu-
azuron (Sigma Chemical Co., St. Louis, MO, USA) was diluted
For the larvae tests, a probit analysis was performed on
in 2% Triton X-100 in technical grade acetone (Merck,
the mortality results using the software Polo-Plus (LeOra
Darmstadt, Germany) to produce a 5% ﬂuazuron solution
Software, 2003). The following parameters were deter-
(50,000 ppm). The 5% ﬂuazuron solution was diluted 1:100
mined for each test: lethal concentrations for 50% (LC50),
in demineralised sterile water to give the top dose solu-
conﬁdence intervals of 95% (CI 95%), and the slope of the
tion for the test: 0.05% (500 ppm) ﬂuazuron in 0.02% Triton
regression line. Resistance ratios (RR) and their CI 95% were
X-100 and 1% acetone. This top dose solution was seri-
generated with the software Polo-Plus using the formula
ally diluted in vehicle (0.02% Triton X-100 in 1% acetone
described by The signiﬁcance of
J. Reck et al. / Veterinary Parasitology 201 (2014) 128–136
Results of the larval tests with cypermethrin, chlorpyriphos, ﬁpronil, amitraz and ivermectin conducted with the Jaguar (resistant) and Porto Alegre (POA,
LC50 (%) (CI 95%)
n, number of larvae; SE, standard error; 2, Chi-square; df, degrees of freedom; LC50, median lethal concentration in % of active ingredient; CI, conﬁdence
interval; RR, resistance ratio.
a Larval packet test.
b Larval immersion test.
each comparison was determined when the calculated con-
supplementary table 1). Treatment efﬁcacy against the
ﬁdence intervals did not overlap.
Jaguar strain was zero at 14, 21 and 28 days p.t.
Additionally, in vitro tests were conducted to charac-
terise ﬂuazuron resistance in the Jaguar strain. The adult
immersion test (AIT) with ﬁve concentrations (0.05, 0.5,
The Jaguar strain showed resistance to all the acari-
5, 50 and 500 ppm) of technical grade ﬂuazuron was per-
cides evaluated in vitro. Results of larval tests are shown in
formed with the POA (susceptible), Jaguar, and Jaguar R
Resistance ratios (RR) were: 31.242 for cyperme-
strains. Fluazuron treatment of the POA strain resulted in a
thrin, 103.926 for chlorpyriphos, 4.441 for ﬁpronil, 11.907
reduction in larval hatch, ranging from 86% at 0.05 ppm to
for amitraz and 3.081 for ivermectin.
99% at 50 and 500 ppm However, ﬂuazuron treat-
Results from the comparison of efﬁcacy under ﬁeld
ment of the Jaguar strain reduced larval hatch by only 21%
conditions between the susceptible SG strain and the
at 0.05 ppm to 52% at 500 ppm (Fluazuron efﬁcacy
Jaguar strain conﬁrmed suspicions by local produc-
was further reduced after selection (using ticks obtained
ers of ﬂuazuron treatment failure. Treatment of cattle
by the selection of ticks from Jaguar strain which survived
experimentally infested with the SG strain (susceptible)
ﬁrst AIT tests), with larval hatch reduced by approximately
resulted in a signiﬁcant reduction in engorged female tick
9% at 0.05 ppm and 20% at 500 ppm
count 14 and 21 days post-treatment (p.t.) respectively
The pattern of ﬂuazuron efﬁcacy upon tick strains using
supplementary table 1). Treatment efﬁcacy in
the artiﬁcial feeding assay (AFA) was similar to the AIT. Off-
cattle experimentally infested with SG strain was 94.09,
spring of the POA strain (susceptible) ticks did not hatch
98.4 and 100% at 14, 21 and 28 days p.t., respectively
when ticks were fed 5 and 10 ppm of ﬂuazuron; only 3%
hatch was observed at 2.5 ppm However, the lar-
Steers in the treated and control groups infested
val hatch of eggs collected from treated Jaguar engorged
with the ticks from the Jaguar strain had equivalent
female ticks was 67% at 2.5 ppm, 43% at 5 ppm, and 8% at
engorged female tick counts through day 28 p.t. (
Fig. 1. Engorged female ticks count from heifers treated with ﬂuazuron under ﬁeld conditions. Panel A, São Gabriel (SG) (susceptible) tick strain infested
heifers. Panel B, Jaguar (resistant) tick strain infested heifers. For both panels, open symbols indicate non-treated heifers and black symbols represent
ﬂuazuron-treated heifers. Results shown are the mean ± S.E.M. of ﬁve animals. Statistical signiﬁcance was analysed by the Student's t test (*P < 0.05,
**P < 0.01).
J. Reck et al. / Veterinary Parasitology 201 (2014) 128–136
Fig. 2. Adult immersion test (AIT) with technical grade ﬂuazuron with the
Porto Alegre (POA) (susceptible, black triangles), Jaguar (resistant, open
squares) and Jaguar R (resistant, open diamonds) strains. Statistical signif-
icance was analysed by one-way analysis of variance (ANOVA) followed by
Tukey's multiple comparison test (***P < 0.001 comparing POA with both
Jaguar and Jaguar R strains; #P < 0.05, ##P < 0.01, ###P < 0.001 comparing
Jaguar and Jaguar R strains).
As a result of the indiscriminate use of acaricides to con-
trol ticks infesting livestock, tick resistance to acaricides
is one of the biggest challenges and a cause of concern
for cattle production wherever R. microplus is endemic or
in areas prone to invasion by this injurious ectoparasite
species. Recently, R. microplus populations with multiple
resistances to acaricides have been identiﬁed in several
ranches, particularly in Brazil and Mexico
Currently, there are six classes of acaricides
available on the Brazilian market to control cattle ticks
with ﬂuazuron being the only AI with no
previous reports of resistance. Here, we report for the ﬁrst
time an R. microplus tick strain that is resistant to ﬂuazuron.
This strain is also the ﬁrst with documented resistance to
Fig. 3. Artiﬁcial feeding assay (AFA) with technical grade ﬂuazuron with
Porto Alegre (POA) (susceptible, open bars) and Jaguar (resistant, black
bars) strains. Statistical signiﬁcance was analysed by one-way analysis of
variance (ANOVA) followed by Tukey's multiple comparison test. Differ-
ent letters above the bars indicate a statistical difference among analysed
groups; P < 0.05.
J. Reck et al. / Veterinary Parasitology 201 (2014) 128–136
the six classes of acaricides used to control cattle tick in
past ﬁeld exposure of tick populations to ﬂuazuron and,
whenever possible, include a comparison between in vitro
In 2010, ticks were collected for the same population
and in vivo results.
from which the Jaguar strain was sourced for the ﬁrst time,
Although the AIT has been questioned as a precise tool
as part of the survey system for cattle tick acaricide resis-
for resistance diagnosis in ticks, particularly for systemic
tance in Rio Grande do Sul state, Brazil, performed by IPVDF.
acaricides it is important to note
At that time, no in vitro test for ﬂuazuron resistance was
that in contrast to ML, a larval test cannot be employed
available, as there was no suspicion of ﬂuazuron resistance.
to investigate ﬂuazuron resistance. This situation is a con-
Subsequently, an investigation concerning ﬂuazuron resis-
sequence of the mechanism of action of ﬂuazuron, which
tance began after ranch employees reported that they could
inhibits chitin synthase activity and subsequently pre-
no longer control ticks infesting cattle even using ﬂuazuron.
vents larval ecdysis As a consequence,
To investigate whether the observation in the ﬁeld was due
ﬂuazuron is a developmental inhibitor and does not kill
to resistance, ticks were sampled and the Jaguar colony was
adult engorged females or larvae as other acaricides do.
established at IPVDF.
Since ﬂuazuron is a systemic acaricide, the artiﬁcial feed-
Results from the ﬁeld trial presented here clearly
ing technique provided a more similar condition to which
demonstrated that ﬂuazuron treatment was unable to
the parasitic life stages are exposed to ﬂuazuron than the
control ticks of the Jaguar strain, neither by reducing
AIT. Pharmacokinetic studies conducted with ﬂuazuron
tick count or larvae hatching. This lack of efﬁcacy was
showed that after the administration of 1.5 mg/kg of live
more noticeable when comparing ﬂuazuron performance
weight, the serum concentration increased to more than
against the SG strain. Field trial studies conducted in South-
1.3 ppm in cattle As the ﬂuazuron label rate
ern Brazil when ﬂuazuron was released on the market
is 2.5 mg/kg of live weight, the concentration of AI added
achieved more than 99% efﬁcacy at 21 days after treat-
to blood fed to ticks in the AFA here ranged from 2.5 to
ment, when using 2 mg/kg of this AI (
10 ppm. Additionally, the marked differences in mortality
In another ﬁeld trial, performed in Australia, the adult tick
after exposure to ﬂuazuron between resistant and suscep-
reduction was 100 and 98% at three and six weeks after
tible tick strains used in this study lend support to the
treatment, respectively Recently, some
resistance status of the Jaguar strain. In this sense, the
authors have shown that ﬂuazuron still gives signiﬁcant
AFA is a technique that is well suited for the characteri-
results under ﬁeld conditions in Brazil.
sation of resistance, especially for systemic drugs with the
in a study using artiﬁcial and natural
understanding that, in some cases, biotransformation of the
tick infestations that ﬂuazuron (2.5 mg/kg) efﬁcacy ranged
parent drug may be required to produce the pharmaco-
from 96% to 100% between 14 and 49 days after treatment.
logically active agent
that ﬂuazuron (2.5 mg/kg) treat-
ment reduced more than 99% of natural tick infestation
Investigation of tick resistance and constant survey of
between 14 and 28 days after treatment.
population susceptibility status are essential to (i) recog-
Scientiﬁc literature concerning the validation and use
nise if acaricide resistance is the cause of tick control
of in vitro tests to evaluate ﬂuazuron susceptibility is lack-
failure; (ii) determine the most efﬁcient acaricide for
ing. This can be attributed to several factors, such as the
each location/ranch; (iii) understand the epidemiology and
absence of a resistant reference strain, and the apparent
spread of resistance; and (iv) develop control strategies
lack of need, since it was assumed that there were no
that minimise the selection of resistant genotypes
cases of ﬁeld resistance to ﬂuazuron. Results from the AIT
RS state, located in Southern Brazil, has a
demonstrated a difference in susceptibility to ﬂuazuron
remarkable history of tick resistance to acaricides as it was
between the POA and Jaguar strains. Moreover, the abil-
the ﬁrst region in Brazil to report resistance to OP and SP,
ity of ﬂuazuron to decrease larval hatch was even lower
and the ﬁrst location in the world to report resistance to
in the Jaguar R selected strain, which indicates there is
ML, mixtures of OP and SP, and in vitro resistance to ﬁpronil
a genetic basis for the trait that could be increased and
selected for in the resistant tick population. Together with
The breeds of cattle, which are
the in vivo results, the AIT has proven to be a useful tool
predominantly Bos taurus taurus, and high stocking rates
to detect resistance to ﬂuazuron in tick populations. Previ-
are two factors that contribute to the problem of acaricide
ously published data showed that a 200 ppm concentration
resistance among populations of R. microplus in RS. These
of ﬂuazuron could be used as a discriminating dose (DD) to
factors lead to heavy tick loads, which, in turn, lead to more
induce total, or near complete inhibition of larvae hatching
treatments per year, and consequently more resistance
in the Parkhurst, Yeerongpilly, and Ulam reference strains
selection. There may be other factors contributing to the
from Australia In our tests, 50 ppm induced
resistance problem in RS requiring further epidemiologi-
more than 99% larvae hatching inhibition in the POA strain,
cal studies for its full identiﬁcation and characterisation.
and less than 50% in the Jaguar strain. Larval hatch in the
In this sense, information regarding resistance in RS could
AIT was close to 50% with the Jaguar strain and >80% with
facilitate the understanding of resistance in other regions of
the Jaguar R strain using ﬂuazuron at 500 ppm. The results
the world where R. microplus impacts livestock production
presented here highlight the need for comprehensive stud-
ies with the AIT and ﬂuazuron, particularly concerning test
The resistance mechanism for ﬂuazuron in the Jaguar
reproducibility and the establishment of a discriminating
strain remains to be fully described. A similar situa-
dose to diagnose resistance. Such studies must consider
tion exists with amitraz, ML, and ﬁpronil. Tick resistance
J. Reck et al. / Veterinary Parasitology 201 (2014) 128–136
against drugs can be due to target site insensitivity and/or
metabolic resistance, including molecular efﬂux pumps.
Studies reported the key role of detoxiﬁcation mecha-
Alonso-Díaz, M.A., Rodríguez-Vivas, R.I., Fragoso-Sánchez, H., Rosario-
nisms in resistance to several drugs (
It is possible that the inten-
Alves-Branco, F.P.J., Sapper, M.F.M., Alves-Branco, L.R.F., Henrique, C.H.,
sive use of different acaricides against the Jaguar strain
Sandoval, G.A.F., Cassol, D.M.S., Mello, I.A.S., Silva, L.M., Toma, S.B.,
Rizzi, V.G., Carneiro, R., 2010.
in the ﬁeld induced several non-speciﬁc pathways which
may contribute to resistance to ﬂuazuron. In fact, syner-
gist bioassays and enzyme activity quantiﬁcation studies
with Musca domestica (
Angus, B.M., 1996.
Lucilia cuprina and Cydia pomonella
(revealed, at least in part, the role
of mixed function oxidase enzymes as a mechanism of
Arteche, C.C.P., 1972.
resistance to diﬂubenzuron, a benzoylphenyl urea com-
pound. The occurrence of chitin synthase alteration which
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as a possible resistance mechanism. These mechanisms
need to be investigated further. Biological phenomena
Bull, M.S., Swindale, S., Overend, D., Hess, E.A., 1996.
underlying tick resistance against ﬂuazuron remain to be
Corrier, D.E., Vizcaino, O., Terry, M., Betancourt, A., Kuttler, K.L., Carson,
Here, we documented the ﬁrst case of ﬂuazuron resis-
C.A., Trevino, G., Ristic, M., 1979.
tance in R. microplus and the ﬁrst tick population resistant
to six classes of acaricide. Assessing the efﬁcacy of ﬂu-
Cuore, U., Trelles, A., Sanchis, J., Gayo, V., Solari, M.A., 2007.
azuron against other populations of R. microplus subjected
to high selection pressure with acaricides and the char-
acterisation of resistance mechanisms in the Jaguar strain
FAO (Food and Agriculture Organization), 1998. Residues of Some
Veterinary Drugs in Animal and Foods, FAO Food and Nutrition
require investigation. This study highlights the spreading
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and emerging issue of multiple resistance to acaricides in
Protection Department, Available on-line at:
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FAO (Food and Agriculture Organization), 2004.
inability to control cattle ticks by using only acaricides cur-
rently available in an indiscriminate manner demonstrates
the need for more research to establish an integrated strat-
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Conﬂict of interest statement
Authors declare no conﬂict of interest.
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handling and AFA, and to Dr. Robert John Miller (ARS, USDA,
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ual de Pesquisa Agropecuária (FEPAGRO), Edital 064/2008
(Conselho Nacional de Desenvolvimento Cientíﬁco e Tec-
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e Abastecimento – MAPA), Projeto SANIMARS (Finan-
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de Amparo à Pesquisa do Estado do Rio Grande do Sul
Guerrero, F.D., Lovis, L., Martins, J.R., 2012.
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Temporally and Regionally Disparate Differences inPlasmin Activity by Tranexamic Acid Daryl L. Reust, MD,* Scott T. Reeves, MD,* James H. Abernathy, III, MD,* Jennifer A. Dixon, MD,‡William F. Gaillard, II, BS,‡ Rupak Mukherjee, PhD,‡ Christine N. Koval, BS,‡ Robert E. Stroud, MS,‡and Francis G. Spinale, MD, PhD†‡ BACKGROUND: A major complication associated with cardiac surgery is excessive and pro-longed bleeding in the perioperative period. Improving coagulation by inhibiting fibrinolysis,primarily through inhibition of plasmin activity (PLact) with antifibrinolytics such as tranexamicacid (TXA), has been a pharmacological mainstay in cardiac surgical patients. Despite its almostubiquitous use, the temporal and regional modulation of PLact profiles by TXA remainsunexplored. Accordingly, we developed a fluorogenic-microdialysis system to measure in vivodynamic changes in PLact after TXA administration in a large animal model.METHODS: Pigs (25–35 kg) were randomly assigned to receive TXA (30 mg/kg, diluted into 50mL normal saline; n ⫽ 9) or vehicle (50 mL normal saline; n ⫽ 7). Microdialysis probes wereplaced in the liver, myocardium, kidney, and quadriceps muscle compartments. The microdialy-sate infusion contained a validated plasmin-specific fluorogenic peptide. The fluorescenceemission (standard fluorogenic units [SFU]) of the interstitial fluid collected from the microdialy-sis probes, which directly reflects PLact, was determined at steady-state baseline and 30, 60,90, and 120 min after TXA/vehicle infusion. Plasma PLact was determined at the same timepoints using the same fluorogenic substrate approach.RESULTS: TXA reduced plasma PLact at 30 min after infusion by ⬎110 SFU compared withvehicle values (P ⬍ 0.05). Specifically, there was a decrease in liver PLact at 90 and 120 minafter TXA infusion of ⬎150 SFU (P ⬍ 0.05) and 175 SFU (P ⬍ 0.05), respectively. The decreasein liver PLact occurred 60 min after the maximal decrease in plasma PLact. In contrast, kidney,heart, and quadriceps PLact transiently increased followed by an overall decrease at 120 min.CONCLUSIONS: Using a large animal model and in vivo microdialysis measurements of PLact,the unique findings from this study were 2-fold. First, TXA induced temporally distinct PLactprofiles within the plasma and selected interstitial compartments. Second, TXA causedregion-specific changes in PLact profiles. These temporal and regional differences in the effectsof TXA may have important therapeutic considerations when managing fibrinolysis in theperioperative period. (Anesth Analg 2010;110:694 –701)
Emergency 3 Contraceptive Pills Key Points for Providers and clients y Emergency contraceptive pills help to prevent pregnancy when taken up to 5 days after unprotected sex. The sooner they are taken, the better. Emergency Contraceptive Pills y Do not disrupt an existing pregnancy.