G Model
ARTICLE IN PRESS
PREVET-3571; No. of Pages 6
Preventive Veterinary Medicine xxx (2014) xxx–xxx
Contents lists available at ScienceDirect
Preventive Veterinary Medicine journal homepage: www.elsevier.com/locate/prevetmed
Efficacy of fipronil (1.0 mg/kg) against Rhipicephalus (Boophilus) microplus strains resistant to ivermectin (0.63 mg/kg) Welber Daniel Zanetti Lopes a,b,∗ , Breno Cayeiro Cruz a , Weslen Fabrício Pires Teixeira a , Gustavo Felippelli a , Willian Giquelin Maciel a , Carolina Buzzulini a , Lucas V. Costa Gomes a , Flávia Favero a , Vando Edésio Soares a,c , Murilo A. Bichuette a , Gilson Pereira de Oliveira a , Alvimar José da Costa a a CPPAR – Animal Health Research Center – Faculdade de Ciências Agrárias e Veterinárias, UNESP, Via de acesso prof. Paulo Donatto Castellani, s/n CEP: 14884-900, Jaboticabal, São Paulo, Brazil b Universidade Federal de Goiás, Faculdade de Medicina Veterinária – Regional de Jataí, Brazil c UNICASTELO – Universidade Camilo Castelo Branco, Campus de Descalvado, Brazil
a r t i c l e
i n f o
Article history: Received 12 January 2014 Received in revised form 6 April 2014 Accepted 17 April 2014
Keywords: Efficacy Fipronil Ivermectin Resistance
a b s t r a c t The present study aimed to evaluate the efficacy of fipronil (1 mg/kg) against three strains of ivermectin-resistant Rhipicephalus (Boophilus) microplus (R. (B.) microplus), naturally infesting cattle from different states of Brazil. Three rural properties with a history of macrocyclic lactones ineffectiveness against the cattle tick, and low frequency use of fipronil in the herd, were selected for the study. The animals were randomized according to the mean tick counts, performed on days −3, −2 and −1, into three groups with 10 animals each: T01, control (saline solution); T02, subcutaneous ivermectin (0.63 mg/kg) and T03, topical fipronil (1 mg/kg). Treatment was performed on day 0. Counts of partially engorged female ticks were performed on days 3, 7 and 14 post-treatment (DPT), and then every 7 days until the 49th DPT. In all three experiments, it was possible to diagnose resistance of R. (B.) microplus to ivermectin (0.63 mg/kg). The maximum efficacy (arithmetic mean) obtained for ivermectin was 64% in experiment II. On the other hand, the formulation containing fipronil (1 mg/kg) reached high efficacy values (≥97%) in all three experiments. The results from all experiments in this study demonstrate the high efficacy of 1 mg/kg fipronil, administered pour-on in naturally infested cattle, against strains of R. (B.) microplus that are resistant to 630 mcg/kg ivermectin. © 2014 Published by Elsevier B.V.
1. Introduction ∗ Corresponding author at: Universidade Federal de Goiás, Faculdade de Medicina Veterinária–Regional de Jataí, Brazil. Tel.: +55 16 32021720; fax: +55 16 32092605. E-mail addresses: [email protected], [email protected] (W.D.Z. Lopes).
Rhipicephalus (Boophilus) microplus (R. (B.) microplus) is a cattle tick and vector of various diseases and, moreover, poses as a major obstacle to animal productivity in tropical and sub-tropical areas of the world where this species is endemic (Thullner, 1997).
http://dx.doi.org/10.1016/j.prevetmed.2014.04.009 0167-5877/© 2014 Published by Elsevier B.V.
Please cite this article in press as: Lopes, W.D.Z., Rhipicephalus (Boophilus) microplus strains resistant http://dx.doi.org/10.1016/j.prevetmed.2014.04.009
et al., Efficacy to ivermectin
of fipronil (1.0 mg/kg) (0.63 mg/kg). PREVET
against (2014),
G Model PREVET-3571; No. of Pages 6
ARTICLE IN PRESS W.D.Z. Lopes et al. / Preventive Veterinary Medicine xxx (2014) xxx–xxx
2
Despite studies on new alternatives (biological control, herbal medicine, vaccines) for the control of R. (B.) microplus, the application of chemical compounds with acaricidal action remains the main choice observed on the field (Lopes et al., 2013). Thus, researchers’ quest for new products that help control this tick continues (Castro-Janer et al., 2010a). In this context, fipronil emerged, a synthetic molecule with insecticidal and acaricidal properties belonging to the phenylpyrazole family (Cole et al., 1993). Initially, fipronil was used to control pests and insects of public health importance (Colliot et al., 1992), later being commercialized against ectoparasites (fleas and ticks) of small animals (Blagburn et al., 1994). Fipronil was subsequently administered to control R. (B.) microplus and the horn fly, Haematobia irritans, on cattle (Davey et al., 1998; Guglielmone et al., 2000; Alberti et al., 2001). Its mechanism of action is based on blocking GABA (gammaaminobutyric acid) action on the pre- and post-synaptic passage of chloride ions through chloride channels (GluCl), which kills the parasites (Zhao et al., 2004). Due to the belief that fipronil acts on the same neurotransmitters (GABA) and chloride channels (GluCl) as the macrocyclic lactones (ML), some researchers have raised the possibility of cross-resistance between phenylpyrazoles (PHPZ) and ML (Guerrero et al., 2012). The present study aimed to evaluate the efficacy of fipronil (1 mg/kg) against ivermectin-resistant strains of R. (B.) microplus in naturally infested cattle from different states of Brazil. 2. Materials and methods 2.1. Study locations, division of groups and efficacy calculations Three experiments were conducted from November 2011 to April 2012. Based on a history of problems with cattle ticks, the ineffectiveness of MLs against this ectoparasite and the consequent absence or low frequency of fipronil use (once per year), three beef cattle properties were selected: two located in the State of Minas Gerais (MG) (“Formiga” in the midwest region and “Andradas” in the southern region) and another in the State of São Paulo (SP) (“Franca” in the northeastern region of the State). According to the owners of the selected farms, the compounds used against R. (B.) microplus are derivates of macrocyclic lactones. The use offluazuron (Acatak® ) and fipronil (Top Line® ) in these farms was performed only four or five times during the 5 years that preceded this study. In each experiment, 30 crossbreed bovines (Bos taurus × Bos indicus) were selected from approximately 50 animals naturally infested with R. (B.) microplus that had not been treated with ecto or endoparasiticides during the previous 90 days. The cattle were allocated to treatment groups on day 0. Animals were randomly designated to treatments according to a completely randomized block design. The block formation was based on the arithmetic mean of female ticks (4.5–8.0 mm long) counted on three consecutive days (−3, −2, −1). In each experiment, the cattle were divided into 10 blocks of three animals each and, inside each block, animals were randomly placed in one of Please cite this article in press as: Lopes, W.D.Z., Rhipicephalus (Boophilus) microplus strains resistant http://dx.doi.org/10.1016/j.prevetmed.2014.04.009
the treatment groups, as represented in Table 1. On day 0 of each experiment, all cattle were weighed to determine the appropriate dose of medication. The number of animals per group used in this study (10 cattle per group) was determined in accordance with recommendations described by the Brazilian Ministry of Agriculture, Livestock and Food Supply (Ministério da Agricultura, Pecuária e Abastecimento – MAPA), Ordinance N◦ 48 (Brasil, 1997). After treatment administration, the pasture that housed the 30 animals was divided into two. Animals belonging to T01 (control) and T02 (ivermectin 0.63 mg/kg, injected through the subcutaneous route – Ivomec Gold® – Merial Animal Health Ltda.) were kept on one side of the pasture, and animals treated with topical fipronil (T03 – 1.0 mg/kg, administered as pour-on – Top Line® – Merial Animal Health Ltda.) were kept on the other side, so that there would be no contamination between groups due to direct contact among animals. The animals had free access to the pasture, water, and mineral supplementation throughout the study. Each animal belonging to T02 received exactly 0.63 mg/kg and cattle belonging to T03 received 1.0 mg/kg, based on the body weight. To evaluate the formulations therapeutic and residual efficacies, tick counts (female with 4.5–8.0 mm long) were performed on days 3, 7, 14 and every 7 days thereafter, until the 49th DPT (Wharton and Utech, 1970). Groups treated with injectable solutions (T01 and T02) were counted first, followed by the group that received topical treatment (T03 – fipronil). Disposable gloves were used and changed between tick counts of each animal. The counts were performed blind. In the present study, all procedures using animals complied with the Ethical Principles in Animal Research adopted by the College of Animal Experimentation (COBEA) and were approved by the Ethical Committee for Animal Welfare of IPESA, Animal Health Research Institute, located on the city of Formiga, state of Minas Gerais, Brazil. 2.2. Data analysis The raw counts of partially engorged female ticks on cattle were log transformed using the equation ln(x + 1) prior to statistical analysis, and underwent UNIVARIATE procedure. Subsequently, all data were analyzed by GLM procedure, using the REPEATED command to test sphericity and orthogonality of the data, which concluded that sphericity condition of the matrix should not be rejected. Differences between treatments were evaluated at a significance level of 5% (P ≤ 0.05). The percentage of efficacy against R. (B.) microplus was calculated using arithmetic means, according to the formula proposed by Roulston et al. (1968) and the Brazilian Ministry of Agriculture, Livestock and Food Supply (Ministério da Agricultura, Pecuária e Abastecimento – MAPA), Ordinance N◦ 48 (Brasil, 1997), described below:
Percentage of efficacy = 1 −
Ta × Cb × 100 Tb × Ca
where Ta is the mean number of partially engorged female ticks counted on treated animals after medication; Tb the mean number of partially engorged female ticks counted on et al., Efficacy to ivermectin
of fipronil (1.0 mg/kg) (0.63 mg/kg). PREVET
against (2014),
G Model
ARTICLE IN PRESS
PREVET-3571; No. of Pages 6
W.D.Z. Lopes et al. / Preventive Veterinary Medicine xxx (2014) xxx–xxx
3
Table 1 Experimental design of experiments I, II and III. Treatment
Animal per treatment/experiment
Group
Route
Posology
T01 T02 T03
10 10 10
Saline solution 0.9% Ivermectin 3.15% Fipronil 1%
Subcutaneous Subcutaneous Pour-on
mL/kg
mg/kg
1/50 1/50 1/10
– 0.63 1.0
Table 2 Mean counts of female R. (B.) microplus (4.5–8.0-mm long) for control and treated cattle groups; efficacy percentages. Arithmetic means. Study I – Formiga, MG, Brazil. Day post treatment
Zeroa 3 7 14 21 28 35 42 49 a b c
Mean [=Slog(x + 1)/n]c of Rhipicephalus (Boophilus) microplus (between 4.5 and 8.0 mm) of cattle
Efficacy (%)
T01: control (saline solution)
T02: ivermectin (0.63 mg/kg)b
T02
T03
Mean
95% CL
Mean
95% CL
Mean
95% CL
3.3A 3.5A 3.5 A 3.5A 3.4A 3.4A 3.4A 3.3A 3.3A
3.1–3.6 3.3–3.7 3.3–3.7 3.3–3.6 3.2–3.6 3.2–3.6 3.2–3.6 3.1–3.5 3.1–3.5
3.3A 3.3A 2.8B 2.6B 3.0B 3.0A 3.0A 3.3A 3.3A
3.1–3.5 3.0–3.5 2.6–3.1 2.3–2.9 2.8–3.3 2.7–3.3 2.8–3.2 3.0–3.5 3.1–3.5
3.3A 2.0B 0.7B 0.6B 0.5B 0.4B 0.9B 2.3B 2.7B
3.1–3.6 1.7–2.3 0.3–1.0 0.2–1.0 0.2–1.0 0.2–0.7 0.5–1.2 1.9–2.8 2.4–3.0
– 13 44 55 28 33 34 3 2
– 78 96 96 97 98 94 58 47
T03: fipronil (1 mg/kg)b
Mean count performed on days −3, −2 and −1. Commercial formulation purchased in the local market. Means values followed by the same letter on the same line do not differ significantly at a 95% confidence interval.
treated animals 3 days before treatment; Ca the mean number of partially engorged female ticks counted on control animals after the experiment started; Cb the mean number of partially engorged ticks counted on control animals 3 days before treatment. 3. Results In experiment I, which took place in the city of Formiga, located in the central-western region of the Minas Gerais state, results showed that ivermectin (0.63 mg/kg) was not effective against R. (B.) microplus. This formulation showed a maximum acaricidal efficacy (arithmetic means) of 55% on the 14th DPT. Thereafter, the efficacy rates of ivermectin
against this ectoparasite declined to 28%, 33%, 34%, 3% and 2% on days 21, 28, 35, 42 and 49 post-treatment, respectively. As for the formulation containing fipronil (1 mg/kg), ≥90% acaricidal efficacy was observed on the 7th DPT (96%), and the efficacy remained above this value until the 35th DPT. The efficacy, then, decreased to 58% and 47% on days 42 and 49, respectively (Table 2). The results of the statistical analysis, regarding the average counts of partially engorged female ticks in each group, are in accordance with previous reports regarding the efficacy of these formulations (Table 2). The mean tick counts in groups treated with ivermectin and fipronil were significantly lower (P ≤ 0.01) when compared to mean counts of the control group, from the 7th to the 21st and the 3rd to
Table 3 Mean counts of female R. (B.) microplus (4.5–8.0-mm long) on control and treated cattle groups; efficacy percentages. Arithmetic means. Study II – Andradas, MG, Brazil. Day post treatment
a
Zero 3 7 14 21 28 35 42 49 a b c
Mean [=Slog(x + 1)/n]c of Rhipicephalus (Boophilus) microplus (between 4.5 and 8.0 mm) of cattle
Efficacy (%)
T01: control (saline solution)
T02: ivermectin (0.63 mg/kg)b
T02
T03
Mean
Mean
– 63 64 62 34 33 2 2 6
95 97 98 99 99 94 78 61
A
3.5 3.3A 3.0A 3.3A 3.4A 3.5A 3.3A 3.4A 3.5A
95% CL 3.1–4.0 2.9–3.7 2.7–3.3 3.1–3.6 3.2–3.6 3.3–3.7 3.1–3.5 3.2–3.6 3.2–3.8
A
3.5 2.4B 1.7B 2.2B 3.0B 3.0B 3.3A 3.3A 3.4A
T03: fipronil (1 mg/kg)b 95% CL
3.2–3.9 2.1–2.8 1.2–2.3 1.7–2.3 2.7–3.2 2.8–3.3 3.2–3.4 2.9–3.7 3.0–3.7
Mean A
3.6 0.7C 0.3C 0.3C 0.1C 0.3C 0.9B 1.9B 2.5B
95% CL 3.2–3.9 0.4–1.1 0.0–0.6 0.0–0.6 0.0–0.4 0.0–0.5 0.7–1.2 1.6–2.2 2.1–2.9
Mean count performed on days −3, −2 and −1. Commercial formulation purchased in the local market. Means values followed by the same letter on the same line do not differ significantly at a 95% confidence interval.
Please cite this article in press as: Lopes, W.D.Z., Rhipicephalus (Boophilus) microplus strains resistant http://dx.doi.org/10.1016/j.prevetmed.2014.04.009
et al., Efficacy to ivermectin
of fipronil (1.0 mg/kg) (0.63 mg/kg). PREVET
against (2014),
G Model
ARTICLE IN PRESS
PREVET-3571; No. of Pages 6
W.D.Z. Lopes et al. / Preventive Veterinary Medicine xxx (2014) xxx–xxx
4
Table 4 Mean counts of female R. (B.) microplus (4.5–8.0-mm long) on control and treated cattle groups; efficacy percentages. Arithmetic means. Study III – Franca, SP, Brazil. Day post treatment
a
Zero 3 7 14 21 28 35 42 49 a b c
Mean [=Slog(x + 1)/n]c of Rhipicephalus (Boophilus) microplus (between 4.5 and 8.0 mm) of cattle
Efficacy (%)
T01: control (saline solution)
T02: ivermectin (0.63 mg/kg)b
T03: fipronil (1 mg/kg)b
T02
Mean
Mean
Mean
A
3.9 4.0A 3.9A 3.6A 3.6A 3.6A 3.2A 3.3A 3.3A
95% CL 3.7–4.2 3.8–4.3 3.5–4.3 3.2–4.0 3.1–4.0 3.3–4.1 2.9–3.5 3.1–3.5 3.1–3.5
A
3.9 3.7A 3.3A 3.5A 3.3A 3.4A 3.0A 2.9A 3.1A
95% CL 3.7–4.1 3.4–3.9 2.8–3.8 3.3–3.6 3.0–3.6 3.1–3.7 2.6–3.3 2.5–3.2 2.7–3.4
A
3.9 0.7B 0.3B 0.5B 0.0B 0.50B 1.0B 1.8B 2.1B
T03
95% CL 3.7–4.2 0.3–1.1 0.0–0.7 0.0–0.9 – 0.0–0.5 0.7–1.3 1.5–2.1 1.8–2.4
– 32 37 26 33 33 17 28 10
97 99 97 100 99 92 79 71
Mean count performed on days −3, −2 and −1. Commercial formulation purchased in the local market. Means values followed by the same letter on the same line do not differ significantly at a 95% confidence interval.
the 49th DPT, respectively. When only T02 and T03 were compared, the mean tick counts of animals that received fipronil were significantly lower (P ≤ 0.01) than the mean counts in ivermectin-treated cattle, throughout the whole post-treatment period (Table 2). When analyzing the results obtained on the second experiment, conducted at a property in the city of Andradas, southern region of MG, it was possible to observe that the general efficacy profiles of both formulations were similar to those obtained in experiment I. Efficacy values between 60% and 65% were achieved by ivermectin from day 3 until day 14 post-treatment, after which those values declined and reached 2%, 2% and 6% efficacy on the 35th, 42nd and 49th DPT, respectively. The topically administered fipronil formulation (1 mg/kg) achieved efficacy values greater than 95% from 3 to 28 days after treatment. On the 35th, 42nd and 49th DPT, this compound demonstrated efficacy values of 94%, 78% and 61%, respectively (Table 3). The groups treated with ivermectin and fipronil had significantly inferior R. (B.) microplus counts (P ≤ 0.01) than control animals. However, fipronil-treated cattle showed significantly fewer ticks (P ≤ 0.01) when compared to animals that received ivermectin, between the 3rd and the 49th DPT (Table 3). Results of the experiment conducted in the city of Franca, northeastern region of São Paulo, further reinforced the findings described in experiments I and II. The degree of R. (B.) microplus population resistance to ivermectin was even more pronounced, and the maximum efficacy obtained by this formulation occurred on the 7th DPT (37%). Between days 3 and 28 post-treatment, the efficacy values remained between 20% and 37%. On the other hand, fipronil achieved high efficacy rates (≥99%), even against an ivermectin-resistant strain of R. (B.) microplus. Table 4 shows that, even on the 3rd day after treatment, fipronil reached 97% acaricidal efficacy against R. (B.) microplus. Values above 99% were obtained on experimental days 21 and 28. After this date, the efficacy results declined to 92%, 79% and 71% on the 35th, 42nd and 49th DPT, respectively (Table 4). Please cite this article in press as: Lopes, W.D.Z., Rhipicephalus (Boophilus) microplus strains resistant http://dx.doi.org/10.1016/j.prevetmed.2014.04.009
There was no significant difference (P > 0.01) in average tick counts between ivermectin-treated and control groups at any time. However, the mean counts of R. (B.) microplus on fipronil-treated cattle were significantly lower (P ≤ 0.01) than the average counts on control animals and those treated with ivermectin throughout the entire post-treatment period (Table 4). 4. Discussion Regarding the protection period of ivermectin (200 or 630 mcg/kg, subcutaneous) against R. (B.) microplus, cattle would have to be treated at maximum intervals of 30 days to ensure no viable ticks could reach repletion and detach from the host (Pereira, 2009; Davey et al., 2010). This treatment interval is dramatically inferior to the label claim for the Long Action ivermectin formulations, which guarantees a 75 days period of prevention against re-infestation. Despite the resistance observed by ivermectin 0.63 mg/kg in this study, the efficacy values reinforce this affirmation described by the above authors, since the efficacy decreased 28 days after treatment. Nowadays, the number of R. (B.) microplus strains resistant to ivermectin is increasing in Brazil (Lopes et al., 2014). In accordance with these authors, the low performance of ivermectin against R. (B.) microplus and helminths may be due to the high frequency of this agent’s use in Brazilian herds. Such increased use has resulted on a larger number of commercial brands available in the Brazilian market for these agents and a subsequent reduction in prices (Sindan, 2008 – Compendium of Products for Veterinary Use). Fipronil (1 mg/kg) has been used to control the horn fly, H. irritans, in Brazilian herds since the early 1990s, and previous studies reported high efficacy in controlling these ectoparasites (Guglielmone et al., 2000; Alberti et al., 2001). In cattle artificially infested with R. (B.) microplus, Davey et al. (1998) reported that the treatment with fipronil 1.0 mg/kg failed to provide complete protection (100%) against larval re-infestation, for the first et al., Efficacy to ivermectin
of fipronil (1.0 mg/kg) (0.63 mg/kg). PREVET
against (2014),
G Model PREVET-3571; No. of Pages 6
ARTICLE IN PRESS W.D.Z. Lopes et al. / Preventive Veterinary Medicine xxx (2014) xxx–xxx
6 weeks after. Using the same methodology, Cuore et al. (2007) reported efficacies ≥90% for fipronil 1.0 mg/kg, during 21 days post-treatment, against this ectoparasite. Efficacy values inferior to 90% were observed by authors of this study, after the 22nd day post-treatment, except on day 24. The results of efficacy, up to 35 days posttreatment, found in this study (≥90%) are similar to those found by Davey et al. (1998) and Cuore et al. (2007). Thus, these results suggest that the cattle would have to be treated at intervals of no more than 35 days apart to ensure that a very small number of ticks can complete their life cycle, but this interval may be inferior on farms with problems of R. (B.) microplus resistant to fipronil. Due to the difficulty associated with conducting studies on the efficacy of acaricidal formulations in naturally infected animals, researchers are developing in vitro methods for efficacy studies of compounds such as ML and PHPZ (Castro-Janer et al., 2009; Klafke et al., 2006). After standardization of these techniques, the first reports of R. (B.) microplus resistance to fipronil emerged from Uruguay and Southern Brazil (Cuore et al., 2007; Castro-Janer et al., 2010a,b, 2011). According to these authors, R. (B.) microplus resistance to fipronil may be facilitated by the use of agricultural fipronil on animals. In Brazil, agricultural fipronil is approximately 20 times cheaper than commercial formulations that contain the same active ingredient and that are available for cattle. For this reason, some livestock producers use agricultural fipronil on animals without knowing the actual dose. This practice can contribute to failures on the control of these ectoparasites as well as animal poisoning. The practice of ignoring the withdraw period, after using the agricultural compound, before marketing products from animal origin, also places human health at risk due to the presence of residues in milk and meat. Additionally, the cost of fipronil for animals in countries such as Uruguay is much smaller than that of the formulations commercially available in Brazil. Therefore, clandestine trade of this active compound between neighboring countries may increase its frequency of use in Brazilian herds, which in turn will cause further increase in the R. (B.) microplus population that is resistant to fipronil (Castro-Janer et al., 2010a). With the emergence of the first fipronil-resistant cattle tick populations, some investigators have hypothesized cross-resistance between PHPZ and ML. Despite being from different families, both types of compounds appear to act on the same neurotransmitter (GABA) and chloride channels (GluCl) (Cole et al., 1993; Durham et al., 2001; Bloomquist, 2003). Among the reports of cattle tick resistance to fipronil, only the work published by Castro-Janer et al. (2011) allows correlation of the in vitro efficacies of fipronil and ivermectin against R. (B.) microplus strains. These researchers evaluated and classified the use history of each drug and observed that in populations treated with ivermectin but not fipronil, larvae were susceptible only to the last compound. Moreover, those populations of larval ticks that were resistant to fipronil showed sensitivity to ivermectin. Maybe, molecular studies will be necessary in future, to assess the possibility of the cross-resistance between these families. Please cite this article in press as: Lopes, W.D.Z., Rhipicephalus (Boophilus) microplus strains resistant http://dx.doi.org/10.1016/j.prevetmed.2014.04.009
5
5. Conclusions It was possible to detect Rhipicephalus (Boophilus) microplus strains resistant to ivermectin, administered subcutaneously at a dosage of 0.63 mg/kg, in three farms, located in different states of Brazil. High efficacy (≥90%) was obtained by fipronil, administered as a pour-on, at the 1.0 mg/kg dose, up to 35 days post-treatment. These results, obtained on all experiments from the present study, demonstrate the high efficacy of 1 mg/kg fipronil, administered pour-on in naturally infested cattle, against strains of Rhipicephalus (Boophilus) microplus that are resistant to 630 mcg/kg ivermectin. References Alberti, H., Alberti, A.L.L., Rinaldi, P.L.F., Lamberti, D.D.G., 2001. Avaliacão da eficácia do fipronil e da deltametrina formulacão “pour-on”, no control da Haematobia irritans, parasitando bovinos em regime de campo, na região oeste do Estado de São Paulo. A Hora Veterinaria 221, 12–13. Blagburn, B.L., Hendrix, C.M., Vaughan, L.L., Lindsay, D.S., 1994. Efficacy of a topical formulation of fipronil against Ctenocephalides felis felis in experimentally infested dogs. In: Proc. 39th Ann. Mtg. Amer. Assoc. Vet. Parasitol., San Francisco, CA, p. 48 pp. Bloomquist, J.R., 2003. Chloride channels as tools for developing selective insecticides. Arch. Insect Biochem. Physiol. 54, 45–156. Brasil, 1997. Ministério da Agricultura e Abastecimento, Secretaria de Defesa Agropecuária, Portaria n.◦ 48, 12/05/1997. Castro-Janer, E., Rifran, L., Piaggio, J., Gil, A., Miller, R.J., Schumaker, T.T., 2009. In vitro tests to establish LC50 and discriminating concentrations for fipronil against Rhipicephalus (Boophilus) microplus (Acari: Ixodidae) and their standardization. Vet. Parasitol. 162, 120–128. Castro-Janer, E., Rifran, L., Gonzalez, P., Piaggio, J., Gill, A., Schumaker, T.T., 2010a. Rhipicephalus (Boophilus) microplus (Acari: Ixodidae) resistance to fipronil in Uruguay evaluated by in vitro bioassays. Vet. Parasitol. 169, 172–177. Castro-Janer, E., Martins, J.R., Mendes, M.C., Namindome, A., Klafke, G.M., Schumaker, T.T., 2010b. Diagnoses of fipronil resistance in Brazilian cattle ticks (Rhipicephalus (Boophilus) microplus) using in vitro larval bioassays. Vet. Parasitol. 173, 300–306. Castro-Janer, E., Rifran, R., Gonzalez, P., Niell, C., Piaggio, J., Gill, A., Schumaker, T.T., 2011. Determination of the susceptibility of Rhipicephalus (Boophilus) microplus (Acari: Ixodidae) to ivermectin and fipronil by Larval Immersion Test (LIT) in Uruguay. Vet. Parasitol. 178, 148–155. Cole, L.M., Nicholson, R.A., Casida, J.E., 1993. Action of phelylpyrazole insecticides at the GABA-gated chloride channel. Pestic. Biochem. Physiol. 46, 47–54. Colliot, F., Kukorowski, K.A., Hawkins, D.W., Roberts, D.A., 1992. A new soil and foliar broad spectrum insecticide. In: Brighton Crop Protection Conference Pests and Diseases, vol. 1, pp. 29–34. Cuore, U., Trelles, A., Sanchís, J., Gayo, V., Solari, M.A., 2007. Primer diagnóstico de resistencia al Fipronil en la garrapata común del ganado Boophilus microplus. Veterinaria (Montevideo) 42, 35–41. Davey, R.B., Ahrens, E.H., George, J.E., Hunter, J.S., Jeannin, P., 1998. Therapeutic and persistent efficacy of fipronil against Boophilus microplus Acari: Ixodidae/on cattle. Vet. Parasitol. 74, 261–276. Davey, R.B., Pound, J.M., Milker, J.A., Klavons, J.A., 2010. Therapeutic and persistent efficacy of a long-acting (LA) formulation of ivermectin against Rhipicephalus (Boophilus) microplus (Acari: Ixodidae) and sera concentration through time in treated cattle. Vet. Parasitol. 169, 149–156. Durham, E.W., Scharf, M.E., Siegfried, B.D., 2001. Toxicity and neurophysiologic effects of fipronil and its oxidative sulfone metabolite on European corn borer larvae (Lepidoptera: Crambidae). Pestic. Biochem. Physiol. 71, 97–106. Guerrero, F.D., Lovis, L., Martins, J.R., 2012. Acaricide resistance mechanisms in Rhipicephalus (Boophilus) microplus. Rev. Bras. Parasitol. Vet. 21, 1–6. Guglielmone, A.A., Volpogni, M.M., Mangold, A.J., Anziani, O.S., 2000. Evaluacio’n de una formulacio’n comercial “pour on” con fipronil al 1%
et al., Efficacy to ivermectin
of fipronil (1.0 mg/kg) (0.63 mg/kg). PREVET
against (2014),
G Model PREVET-3571; No. of Pages 6
6
ARTICLE IN PRESS W.D.Z. Lopes et al. / Preventive Veterinary Medicine xxx (2014) xxx–xxx
para el control de Haematobia irritans en vaquillonas Holando naturalmente infestadas. Veterinaria (Argentina) 17, 108–113. Klafke, G.M., Sabatini, G.A., de Albuquerque, T.A., Martins, J.R., Kemp, D.H., Miller, R.J., Schumaker, T.T., 2006. Larval immersion tests with ivermectin in populations of the cattle tick Rhipicephalus (Boophilus) microplus (Acari: Ixodidae) from State of Sao Paulo, Brazil. Vet. Parasitol. 142, 386–390. Lopes, W.D.Z., Teixeira, W.F.P., Matos, L.V.S., Felippelli, G., Cruz, B.C., Maciel, W.G., Buzzulini, C., Fávero, F.C., Soares, V.E., Oliveira, G.P., Costa, A.J., 2013. Effects of macrocyclic lactones on the reproductive parameters of engorged Rhipicephalus (Boophilus) microplus females detached from experimentally infested cattle. Exp. Parasitol. 135, 72–78. Lopes, W.D.Z., Teixeira, W.F.P., Felippelli, G., Cruz, B.C., Maciel, W.G., Soares, V.E., Santos, T.R., Matos, L., Favero, F., Costa, A.J., 2014. Assessing resistance of ivermectin and moxidectin against nematodes in cattle naturally infected using three different methodologies. Res. Vet. Sci. 96, 133–138.
Please cite this article in press as: Lopes, W.D.Z., Rhipicephalus (Boophilus) microplus strains resistant http://dx.doi.org/10.1016/j.prevetmed.2014.04.009
Pereira, J.R., 2009. The efficiency of avermectins (abamectin, doramectin and ivermectin) in the control of Boophilus microplus, in artificially infested bovines kept in field conditions. Vet. Parasitol. 162, 116–119. Roulston, W.J., Wharton, R.H., Nolam, J., Kevr, J.D., 1968. Acaricide tests on the bjarra strain of organophosphorus resistant cattle ticks Boophilus microplus from southern Queensland. Aust. Vet. J. 43, 129–134. Sindicato Nacional da Indústria de Produtos para Saúde Animal – SINDAN, Mercado Veterinário, São Paulo, 2008. Thullner, F., 1997. Impact of pesticide resistance and network for global pesticide resistance management based on a regional structure. WARRMZ 89, 41–47. Wharton, R.H., Utech, K.B.W., 1970. Relation between engorgement and dropping of Boophilus microplus to assessment of tick number in cattle. Aust. Entomol. Soc. 9, 171–182. Zhao, X., Yeh, J.Z., Salgado, V.L., Narahashi, T., 2004. Fipronil is a potent open channel blocker of glutamate-activated chloride channels in cockroach neurons. J. Pharmacol. Exp. Ther. 310, 192–201.
et al., Efficacy to ivermectin
of fipronil (1.0 mg/kg) (0.63 mg/kg). PREVET
against (2014),
ARTICLE IN PRESS
PREVET-3571; No. of Pages 6
Preventive Veterinary Medicine xxx (2014) xxx–xxx
Contents lists available at ScienceDirect
Preventive Veterinary Medicine journal homepage: www.elsevier.com/locate/prevetmed
Efficacy of fipronil (1.0 mg/kg) against Rhipicephalus (Boophilus) microplus strains resistant to ivermectin (0.63 mg/kg) Welber Daniel Zanetti Lopes a,b,∗ , Breno Cayeiro Cruz a , Weslen Fabrício Pires Teixeira a , Gustavo Felippelli a , Willian Giquelin Maciel a , Carolina Buzzulini a , Lucas V. Costa Gomes a , Flávia Favero a , Vando Edésio Soares a,c , Murilo A. Bichuette a , Gilson Pereira de Oliveira a , Alvimar José da Costa a a CPPAR – Animal Health Research Center – Faculdade de Ciências Agrárias e Veterinárias, UNESP, Via de acesso prof. Paulo Donatto Castellani, s/n CEP: 14884-900, Jaboticabal, São Paulo, Brazil b Universidade Federal de Goiás, Faculdade de Medicina Veterinária – Regional de Jataí, Brazil c UNICASTELO – Universidade Camilo Castelo Branco, Campus de Descalvado, Brazil
a r t i c l e
i n f o
Article history: Received 12 January 2014 Received in revised form 6 April 2014 Accepted 17 April 2014
Keywords: Efficacy Fipronil Ivermectin Resistance
a b s t r a c t The present study aimed to evaluate the efficacy of fipronil (1 mg/kg) against three strains of ivermectin-resistant Rhipicephalus (Boophilus) microplus (R. (B.) microplus), naturally infesting cattle from different states of Brazil. Three rural properties with a history of macrocyclic lactones ineffectiveness against the cattle tick, and low frequency use of fipronil in the herd, were selected for the study. The animals were randomized according to the mean tick counts, performed on days −3, −2 and −1, into three groups with 10 animals each: T01, control (saline solution); T02, subcutaneous ivermectin (0.63 mg/kg) and T03, topical fipronil (1 mg/kg). Treatment was performed on day 0. Counts of partially engorged female ticks were performed on days 3, 7 and 14 post-treatment (DPT), and then every 7 days until the 49th DPT. In all three experiments, it was possible to diagnose resistance of R. (B.) microplus to ivermectin (0.63 mg/kg). The maximum efficacy (arithmetic mean) obtained for ivermectin was 64% in experiment II. On the other hand, the formulation containing fipronil (1 mg/kg) reached high efficacy values (≥97%) in all three experiments. The results from all experiments in this study demonstrate the high efficacy of 1 mg/kg fipronil, administered pour-on in naturally infested cattle, against strains of R. (B.) microplus that are resistant to 630 mcg/kg ivermectin. © 2014 Published by Elsevier B.V.
1. Introduction ∗ Corresponding author at: Universidade Federal de Goiás, Faculdade de Medicina Veterinária–Regional de Jataí, Brazil. Tel.: +55 16 32021720; fax: +55 16 32092605. E-mail addresses: [email protected], [email protected] (W.D.Z. Lopes).
Rhipicephalus (Boophilus) microplus (R. (B.) microplus) is a cattle tick and vector of various diseases and, moreover, poses as a major obstacle to animal productivity in tropical and sub-tropical areas of the world where this species is endemic (Thullner, 1997).
http://dx.doi.org/10.1016/j.prevetmed.2014.04.009 0167-5877/© 2014 Published by Elsevier B.V.
Please cite this article in press as: Lopes, W.D.Z., Rhipicephalus (Boophilus) microplus strains resistant http://dx.doi.org/10.1016/j.prevetmed.2014.04.009
et al., Efficacy to ivermectin
of fipronil (1.0 mg/kg) (0.63 mg/kg). PREVET
against (2014),
G Model PREVET-3571; No. of Pages 6
ARTICLE IN PRESS W.D.Z. Lopes et al. / Preventive Veterinary Medicine xxx (2014) xxx–xxx
2
Despite studies on new alternatives (biological control, herbal medicine, vaccines) for the control of R. (B.) microplus, the application of chemical compounds with acaricidal action remains the main choice observed on the field (Lopes et al., 2013). Thus, researchers’ quest for new products that help control this tick continues (Castro-Janer et al., 2010a). In this context, fipronil emerged, a synthetic molecule with insecticidal and acaricidal properties belonging to the phenylpyrazole family (Cole et al., 1993). Initially, fipronil was used to control pests and insects of public health importance (Colliot et al., 1992), later being commercialized against ectoparasites (fleas and ticks) of small animals (Blagburn et al., 1994). Fipronil was subsequently administered to control R. (B.) microplus and the horn fly, Haematobia irritans, on cattle (Davey et al., 1998; Guglielmone et al., 2000; Alberti et al., 2001). Its mechanism of action is based on blocking GABA (gammaaminobutyric acid) action on the pre- and post-synaptic passage of chloride ions through chloride channels (GluCl), which kills the parasites (Zhao et al., 2004). Due to the belief that fipronil acts on the same neurotransmitters (GABA) and chloride channels (GluCl) as the macrocyclic lactones (ML), some researchers have raised the possibility of cross-resistance between phenylpyrazoles (PHPZ) and ML (Guerrero et al., 2012). The present study aimed to evaluate the efficacy of fipronil (1 mg/kg) against ivermectin-resistant strains of R. (B.) microplus in naturally infested cattle from different states of Brazil. 2. Materials and methods 2.1. Study locations, division of groups and efficacy calculations Three experiments were conducted from November 2011 to April 2012. Based on a history of problems with cattle ticks, the ineffectiveness of MLs against this ectoparasite and the consequent absence or low frequency of fipronil use (once per year), three beef cattle properties were selected: two located in the State of Minas Gerais (MG) (“Formiga” in the midwest region and “Andradas” in the southern region) and another in the State of São Paulo (SP) (“Franca” in the northeastern region of the State). According to the owners of the selected farms, the compounds used against R. (B.) microplus are derivates of macrocyclic lactones. The use offluazuron (Acatak® ) and fipronil (Top Line® ) in these farms was performed only four or five times during the 5 years that preceded this study. In each experiment, 30 crossbreed bovines (Bos taurus × Bos indicus) were selected from approximately 50 animals naturally infested with R. (B.) microplus that had not been treated with ecto or endoparasiticides during the previous 90 days. The cattle were allocated to treatment groups on day 0. Animals were randomly designated to treatments according to a completely randomized block design. The block formation was based on the arithmetic mean of female ticks (4.5–8.0 mm long) counted on three consecutive days (−3, −2, −1). In each experiment, the cattle were divided into 10 blocks of three animals each and, inside each block, animals were randomly placed in one of Please cite this article in press as: Lopes, W.D.Z., Rhipicephalus (Boophilus) microplus strains resistant http://dx.doi.org/10.1016/j.prevetmed.2014.04.009
the treatment groups, as represented in Table 1. On day 0 of each experiment, all cattle were weighed to determine the appropriate dose of medication. The number of animals per group used in this study (10 cattle per group) was determined in accordance with recommendations described by the Brazilian Ministry of Agriculture, Livestock and Food Supply (Ministério da Agricultura, Pecuária e Abastecimento – MAPA), Ordinance N◦ 48 (Brasil, 1997). After treatment administration, the pasture that housed the 30 animals was divided into two. Animals belonging to T01 (control) and T02 (ivermectin 0.63 mg/kg, injected through the subcutaneous route – Ivomec Gold® – Merial Animal Health Ltda.) were kept on one side of the pasture, and animals treated with topical fipronil (T03 – 1.0 mg/kg, administered as pour-on – Top Line® – Merial Animal Health Ltda.) were kept on the other side, so that there would be no contamination between groups due to direct contact among animals. The animals had free access to the pasture, water, and mineral supplementation throughout the study. Each animal belonging to T02 received exactly 0.63 mg/kg and cattle belonging to T03 received 1.0 mg/kg, based on the body weight. To evaluate the formulations therapeutic and residual efficacies, tick counts (female with 4.5–8.0 mm long) were performed on days 3, 7, 14 and every 7 days thereafter, until the 49th DPT (Wharton and Utech, 1970). Groups treated with injectable solutions (T01 and T02) were counted first, followed by the group that received topical treatment (T03 – fipronil). Disposable gloves were used and changed between tick counts of each animal. The counts were performed blind. In the present study, all procedures using animals complied with the Ethical Principles in Animal Research adopted by the College of Animal Experimentation (COBEA) and were approved by the Ethical Committee for Animal Welfare of IPESA, Animal Health Research Institute, located on the city of Formiga, state of Minas Gerais, Brazil. 2.2. Data analysis The raw counts of partially engorged female ticks on cattle were log transformed using the equation ln(x + 1) prior to statistical analysis, and underwent UNIVARIATE procedure. Subsequently, all data were analyzed by GLM procedure, using the REPEATED command to test sphericity and orthogonality of the data, which concluded that sphericity condition of the matrix should not be rejected. Differences between treatments were evaluated at a significance level of 5% (P ≤ 0.05). The percentage of efficacy against R. (B.) microplus was calculated using arithmetic means, according to the formula proposed by Roulston et al. (1968) and the Brazilian Ministry of Agriculture, Livestock and Food Supply (Ministério da Agricultura, Pecuária e Abastecimento – MAPA), Ordinance N◦ 48 (Brasil, 1997), described below:
Percentage of efficacy = 1 −
Ta × Cb × 100 Tb × Ca
where Ta is the mean number of partially engorged female ticks counted on treated animals after medication; Tb the mean number of partially engorged female ticks counted on et al., Efficacy to ivermectin
of fipronil (1.0 mg/kg) (0.63 mg/kg). PREVET
against (2014),
G Model
ARTICLE IN PRESS
PREVET-3571; No. of Pages 6
W.D.Z. Lopes et al. / Preventive Veterinary Medicine xxx (2014) xxx–xxx
3
Table 1 Experimental design of experiments I, II and III. Treatment
Animal per treatment/experiment
Group
Route
Posology
T01 T02 T03
10 10 10
Saline solution 0.9% Ivermectin 3.15% Fipronil 1%
Subcutaneous Subcutaneous Pour-on
mL/kg
mg/kg
1/50 1/50 1/10
– 0.63 1.0
Table 2 Mean counts of female R. (B.) microplus (4.5–8.0-mm long) for control and treated cattle groups; efficacy percentages. Arithmetic means. Study I – Formiga, MG, Brazil. Day post treatment
Zeroa 3 7 14 21 28 35 42 49 a b c
Mean [=Slog(x + 1)/n]c of Rhipicephalus (Boophilus) microplus (between 4.5 and 8.0 mm) of cattle
Efficacy (%)
T01: control (saline solution)
T02: ivermectin (0.63 mg/kg)b
T02
T03
Mean
95% CL
Mean
95% CL
Mean
95% CL
3.3A 3.5A 3.5 A 3.5A 3.4A 3.4A 3.4A 3.3A 3.3A
3.1–3.6 3.3–3.7 3.3–3.7 3.3–3.6 3.2–3.6 3.2–3.6 3.2–3.6 3.1–3.5 3.1–3.5
3.3A 3.3A 2.8B 2.6B 3.0B 3.0A 3.0A 3.3A 3.3A
3.1–3.5 3.0–3.5 2.6–3.1 2.3–2.9 2.8–3.3 2.7–3.3 2.8–3.2 3.0–3.5 3.1–3.5
3.3A 2.0B 0.7B 0.6B 0.5B 0.4B 0.9B 2.3B 2.7B
3.1–3.6 1.7–2.3 0.3–1.0 0.2–1.0 0.2–1.0 0.2–0.7 0.5–1.2 1.9–2.8 2.4–3.0
– 13 44 55 28 33 34 3 2
– 78 96 96 97 98 94 58 47
T03: fipronil (1 mg/kg)b
Mean count performed on days −3, −2 and −1. Commercial formulation purchased in the local market. Means values followed by the same letter on the same line do not differ significantly at a 95% confidence interval.
treated animals 3 days before treatment; Ca the mean number of partially engorged female ticks counted on control animals after the experiment started; Cb the mean number of partially engorged ticks counted on control animals 3 days before treatment. 3. Results In experiment I, which took place in the city of Formiga, located in the central-western region of the Minas Gerais state, results showed that ivermectin (0.63 mg/kg) was not effective against R. (B.) microplus. This formulation showed a maximum acaricidal efficacy (arithmetic means) of 55% on the 14th DPT. Thereafter, the efficacy rates of ivermectin
against this ectoparasite declined to 28%, 33%, 34%, 3% and 2% on days 21, 28, 35, 42 and 49 post-treatment, respectively. As for the formulation containing fipronil (1 mg/kg), ≥90% acaricidal efficacy was observed on the 7th DPT (96%), and the efficacy remained above this value until the 35th DPT. The efficacy, then, decreased to 58% and 47% on days 42 and 49, respectively (Table 2). The results of the statistical analysis, regarding the average counts of partially engorged female ticks in each group, are in accordance with previous reports regarding the efficacy of these formulations (Table 2). The mean tick counts in groups treated with ivermectin and fipronil were significantly lower (P ≤ 0.01) when compared to mean counts of the control group, from the 7th to the 21st and the 3rd to
Table 3 Mean counts of female R. (B.) microplus (4.5–8.0-mm long) on control and treated cattle groups; efficacy percentages. Arithmetic means. Study II – Andradas, MG, Brazil. Day post treatment
a
Zero 3 7 14 21 28 35 42 49 a b c
Mean [=Slog(x + 1)/n]c of Rhipicephalus (Boophilus) microplus (between 4.5 and 8.0 mm) of cattle
Efficacy (%)
T01: control (saline solution)
T02: ivermectin (0.63 mg/kg)b
T02
T03
Mean
Mean
– 63 64 62 34 33 2 2 6
95 97 98 99 99 94 78 61
A
3.5 3.3A 3.0A 3.3A 3.4A 3.5A 3.3A 3.4A 3.5A
95% CL 3.1–4.0 2.9–3.7 2.7–3.3 3.1–3.6 3.2–3.6 3.3–3.7 3.1–3.5 3.2–3.6 3.2–3.8
A
3.5 2.4B 1.7B 2.2B 3.0B 3.0B 3.3A 3.3A 3.4A
T03: fipronil (1 mg/kg)b 95% CL
3.2–3.9 2.1–2.8 1.2–2.3 1.7–2.3 2.7–3.2 2.8–3.3 3.2–3.4 2.9–3.7 3.0–3.7
Mean A
3.6 0.7C 0.3C 0.3C 0.1C 0.3C 0.9B 1.9B 2.5B
95% CL 3.2–3.9 0.4–1.1 0.0–0.6 0.0–0.6 0.0–0.4 0.0–0.5 0.7–1.2 1.6–2.2 2.1–2.9
Mean count performed on days −3, −2 and −1. Commercial formulation purchased in the local market. Means values followed by the same letter on the same line do not differ significantly at a 95% confidence interval.
Please cite this article in press as: Lopes, W.D.Z., Rhipicephalus (Boophilus) microplus strains resistant http://dx.doi.org/10.1016/j.prevetmed.2014.04.009
et al., Efficacy to ivermectin
of fipronil (1.0 mg/kg) (0.63 mg/kg). PREVET
against (2014),
G Model
ARTICLE IN PRESS
PREVET-3571; No. of Pages 6
W.D.Z. Lopes et al. / Preventive Veterinary Medicine xxx (2014) xxx–xxx
4
Table 4 Mean counts of female R. (B.) microplus (4.5–8.0-mm long) on control and treated cattle groups; efficacy percentages. Arithmetic means. Study III – Franca, SP, Brazil. Day post treatment
a
Zero 3 7 14 21 28 35 42 49 a b c
Mean [=Slog(x + 1)/n]c of Rhipicephalus (Boophilus) microplus (between 4.5 and 8.0 mm) of cattle
Efficacy (%)
T01: control (saline solution)
T02: ivermectin (0.63 mg/kg)b
T03: fipronil (1 mg/kg)b
T02
Mean
Mean
Mean
A
3.9 4.0A 3.9A 3.6A 3.6A 3.6A 3.2A 3.3A 3.3A
95% CL 3.7–4.2 3.8–4.3 3.5–4.3 3.2–4.0 3.1–4.0 3.3–4.1 2.9–3.5 3.1–3.5 3.1–3.5
A
3.9 3.7A 3.3A 3.5A 3.3A 3.4A 3.0A 2.9A 3.1A
95% CL 3.7–4.1 3.4–3.9 2.8–3.8 3.3–3.6 3.0–3.6 3.1–3.7 2.6–3.3 2.5–3.2 2.7–3.4
A
3.9 0.7B 0.3B 0.5B 0.0B 0.50B 1.0B 1.8B 2.1B
T03
95% CL 3.7–4.2 0.3–1.1 0.0–0.7 0.0–0.9 – 0.0–0.5 0.7–1.3 1.5–2.1 1.8–2.4
– 32 37 26 33 33 17 28 10
97 99 97 100 99 92 79 71
Mean count performed on days −3, −2 and −1. Commercial formulation purchased in the local market. Means values followed by the same letter on the same line do not differ significantly at a 95% confidence interval.
the 49th DPT, respectively. When only T02 and T03 were compared, the mean tick counts of animals that received fipronil were significantly lower (P ≤ 0.01) than the mean counts in ivermectin-treated cattle, throughout the whole post-treatment period (Table 2). When analyzing the results obtained on the second experiment, conducted at a property in the city of Andradas, southern region of MG, it was possible to observe that the general efficacy profiles of both formulations were similar to those obtained in experiment I. Efficacy values between 60% and 65% were achieved by ivermectin from day 3 until day 14 post-treatment, after which those values declined and reached 2%, 2% and 6% efficacy on the 35th, 42nd and 49th DPT, respectively. The topically administered fipronil formulation (1 mg/kg) achieved efficacy values greater than 95% from 3 to 28 days after treatment. On the 35th, 42nd and 49th DPT, this compound demonstrated efficacy values of 94%, 78% and 61%, respectively (Table 3). The groups treated with ivermectin and fipronil had significantly inferior R. (B.) microplus counts (P ≤ 0.01) than control animals. However, fipronil-treated cattle showed significantly fewer ticks (P ≤ 0.01) when compared to animals that received ivermectin, between the 3rd and the 49th DPT (Table 3). Results of the experiment conducted in the city of Franca, northeastern region of São Paulo, further reinforced the findings described in experiments I and II. The degree of R. (B.) microplus population resistance to ivermectin was even more pronounced, and the maximum efficacy obtained by this formulation occurred on the 7th DPT (37%). Between days 3 and 28 post-treatment, the efficacy values remained between 20% and 37%. On the other hand, fipronil achieved high efficacy rates (≥99%), even against an ivermectin-resistant strain of R. (B.) microplus. Table 4 shows that, even on the 3rd day after treatment, fipronil reached 97% acaricidal efficacy against R. (B.) microplus. Values above 99% were obtained on experimental days 21 and 28. After this date, the efficacy results declined to 92%, 79% and 71% on the 35th, 42nd and 49th DPT, respectively (Table 4). Please cite this article in press as: Lopes, W.D.Z., Rhipicephalus (Boophilus) microplus strains resistant http://dx.doi.org/10.1016/j.prevetmed.2014.04.009
There was no significant difference (P > 0.01) in average tick counts between ivermectin-treated and control groups at any time. However, the mean counts of R. (B.) microplus on fipronil-treated cattle were significantly lower (P ≤ 0.01) than the average counts on control animals and those treated with ivermectin throughout the entire post-treatment period (Table 4). 4. Discussion Regarding the protection period of ivermectin (200 or 630 mcg/kg, subcutaneous) against R. (B.) microplus, cattle would have to be treated at maximum intervals of 30 days to ensure no viable ticks could reach repletion and detach from the host (Pereira, 2009; Davey et al., 2010). This treatment interval is dramatically inferior to the label claim for the Long Action ivermectin formulations, which guarantees a 75 days period of prevention against re-infestation. Despite the resistance observed by ivermectin 0.63 mg/kg in this study, the efficacy values reinforce this affirmation described by the above authors, since the efficacy decreased 28 days after treatment. Nowadays, the number of R. (B.) microplus strains resistant to ivermectin is increasing in Brazil (Lopes et al., 2014). In accordance with these authors, the low performance of ivermectin against R. (B.) microplus and helminths may be due to the high frequency of this agent’s use in Brazilian herds. Such increased use has resulted on a larger number of commercial brands available in the Brazilian market for these agents and a subsequent reduction in prices (Sindan, 2008 – Compendium of Products for Veterinary Use). Fipronil (1 mg/kg) has been used to control the horn fly, H. irritans, in Brazilian herds since the early 1990s, and previous studies reported high efficacy in controlling these ectoparasites (Guglielmone et al., 2000; Alberti et al., 2001). In cattle artificially infested with R. (B.) microplus, Davey et al. (1998) reported that the treatment with fipronil 1.0 mg/kg failed to provide complete protection (100%) against larval re-infestation, for the first et al., Efficacy to ivermectin
of fipronil (1.0 mg/kg) (0.63 mg/kg). PREVET
against (2014),
G Model PREVET-3571; No. of Pages 6
ARTICLE IN PRESS W.D.Z. Lopes et al. / Preventive Veterinary Medicine xxx (2014) xxx–xxx
6 weeks after. Using the same methodology, Cuore et al. (2007) reported efficacies ≥90% for fipronil 1.0 mg/kg, during 21 days post-treatment, against this ectoparasite. Efficacy values inferior to 90% were observed by authors of this study, after the 22nd day post-treatment, except on day 24. The results of efficacy, up to 35 days posttreatment, found in this study (≥90%) are similar to those found by Davey et al. (1998) and Cuore et al. (2007). Thus, these results suggest that the cattle would have to be treated at intervals of no more than 35 days apart to ensure that a very small number of ticks can complete their life cycle, but this interval may be inferior on farms with problems of R. (B.) microplus resistant to fipronil. Due to the difficulty associated with conducting studies on the efficacy of acaricidal formulations in naturally infected animals, researchers are developing in vitro methods for efficacy studies of compounds such as ML and PHPZ (Castro-Janer et al., 2009; Klafke et al., 2006). After standardization of these techniques, the first reports of R. (B.) microplus resistance to fipronil emerged from Uruguay and Southern Brazil (Cuore et al., 2007; Castro-Janer et al., 2010a,b, 2011). According to these authors, R. (B.) microplus resistance to fipronil may be facilitated by the use of agricultural fipronil on animals. In Brazil, agricultural fipronil is approximately 20 times cheaper than commercial formulations that contain the same active ingredient and that are available for cattle. For this reason, some livestock producers use agricultural fipronil on animals without knowing the actual dose. This practice can contribute to failures on the control of these ectoparasites as well as animal poisoning. The practice of ignoring the withdraw period, after using the agricultural compound, before marketing products from animal origin, also places human health at risk due to the presence of residues in milk and meat. Additionally, the cost of fipronil for animals in countries such as Uruguay is much smaller than that of the formulations commercially available in Brazil. Therefore, clandestine trade of this active compound between neighboring countries may increase its frequency of use in Brazilian herds, which in turn will cause further increase in the R. (B.) microplus population that is resistant to fipronil (Castro-Janer et al., 2010a). With the emergence of the first fipronil-resistant cattle tick populations, some investigators have hypothesized cross-resistance between PHPZ and ML. Despite being from different families, both types of compounds appear to act on the same neurotransmitter (GABA) and chloride channels (GluCl) (Cole et al., 1993; Durham et al., 2001; Bloomquist, 2003). Among the reports of cattle tick resistance to fipronil, only the work published by Castro-Janer et al. (2011) allows correlation of the in vitro efficacies of fipronil and ivermectin against R. (B.) microplus strains. These researchers evaluated and classified the use history of each drug and observed that in populations treated with ivermectin but not fipronil, larvae were susceptible only to the last compound. Moreover, those populations of larval ticks that were resistant to fipronil showed sensitivity to ivermectin. Maybe, molecular studies will be necessary in future, to assess the possibility of the cross-resistance between these families. Please cite this article in press as: Lopes, W.D.Z., Rhipicephalus (Boophilus) microplus strains resistant http://dx.doi.org/10.1016/j.prevetmed.2014.04.009
5
5. Conclusions It was possible to detect Rhipicephalus (Boophilus) microplus strains resistant to ivermectin, administered subcutaneously at a dosage of 0.63 mg/kg, in three farms, located in different states of Brazil. High efficacy (≥90%) was obtained by fipronil, administered as a pour-on, at the 1.0 mg/kg dose, up to 35 days post-treatment. These results, obtained on all experiments from the present study, demonstrate the high efficacy of 1 mg/kg fipronil, administered pour-on in naturally infested cattle, against strains of Rhipicephalus (Boophilus) microplus that are resistant to 630 mcg/kg ivermectin. References Alberti, H., Alberti, A.L.L., Rinaldi, P.L.F., Lamberti, D.D.G., 2001. Avaliacão da eficácia do fipronil e da deltametrina formulacão “pour-on”, no control da Haematobia irritans, parasitando bovinos em regime de campo, na região oeste do Estado de São Paulo. A Hora Veterinaria 221, 12–13. Blagburn, B.L., Hendrix, C.M., Vaughan, L.L., Lindsay, D.S., 1994. Efficacy of a topical formulation of fipronil against Ctenocephalides felis felis in experimentally infested dogs. In: Proc. 39th Ann. Mtg. Amer. Assoc. Vet. Parasitol., San Francisco, CA, p. 48 pp. Bloomquist, J.R., 2003. Chloride channels as tools for developing selective insecticides. Arch. Insect Biochem. Physiol. 54, 45–156. Brasil, 1997. Ministério da Agricultura e Abastecimento, Secretaria de Defesa Agropecuária, Portaria n.◦ 48, 12/05/1997. Castro-Janer, E., Rifran, L., Piaggio, J., Gil, A., Miller, R.J., Schumaker, T.T., 2009. In vitro tests to establish LC50 and discriminating concentrations for fipronil against Rhipicephalus (Boophilus) microplus (Acari: Ixodidae) and their standardization. Vet. Parasitol. 162, 120–128. Castro-Janer, E., Rifran, L., Gonzalez, P., Piaggio, J., Gill, A., Schumaker, T.T., 2010a. Rhipicephalus (Boophilus) microplus (Acari: Ixodidae) resistance to fipronil in Uruguay evaluated by in vitro bioassays. Vet. Parasitol. 169, 172–177. Castro-Janer, E., Martins, J.R., Mendes, M.C., Namindome, A., Klafke, G.M., Schumaker, T.T., 2010b. Diagnoses of fipronil resistance in Brazilian cattle ticks (Rhipicephalus (Boophilus) microplus) using in vitro larval bioassays. Vet. Parasitol. 173, 300–306. Castro-Janer, E., Rifran, R., Gonzalez, P., Niell, C., Piaggio, J., Gill, A., Schumaker, T.T., 2011. Determination of the susceptibility of Rhipicephalus (Boophilus) microplus (Acari: Ixodidae) to ivermectin and fipronil by Larval Immersion Test (LIT) in Uruguay. Vet. Parasitol. 178, 148–155. Cole, L.M., Nicholson, R.A., Casida, J.E., 1993. Action of phelylpyrazole insecticides at the GABA-gated chloride channel. Pestic. Biochem. Physiol. 46, 47–54. Colliot, F., Kukorowski, K.A., Hawkins, D.W., Roberts, D.A., 1992. A new soil and foliar broad spectrum insecticide. In: Brighton Crop Protection Conference Pests and Diseases, vol. 1, pp. 29–34. Cuore, U., Trelles, A., Sanchís, J., Gayo, V., Solari, M.A., 2007. Primer diagnóstico de resistencia al Fipronil en la garrapata común del ganado Boophilus microplus. Veterinaria (Montevideo) 42, 35–41. Davey, R.B., Ahrens, E.H., George, J.E., Hunter, J.S., Jeannin, P., 1998. Therapeutic and persistent efficacy of fipronil against Boophilus microplus Acari: Ixodidae/on cattle. Vet. Parasitol. 74, 261–276. Davey, R.B., Pound, J.M., Milker, J.A., Klavons, J.A., 2010. Therapeutic and persistent efficacy of a long-acting (LA) formulation of ivermectin against Rhipicephalus (Boophilus) microplus (Acari: Ixodidae) and sera concentration through time in treated cattle. Vet. Parasitol. 169, 149–156. Durham, E.W., Scharf, M.E., Siegfried, B.D., 2001. Toxicity and neurophysiologic effects of fipronil and its oxidative sulfone metabolite on European corn borer larvae (Lepidoptera: Crambidae). Pestic. Biochem. Physiol. 71, 97–106. Guerrero, F.D., Lovis, L., Martins, J.R., 2012. Acaricide resistance mechanisms in Rhipicephalus (Boophilus) microplus. Rev. Bras. Parasitol. Vet. 21, 1–6. Guglielmone, A.A., Volpogni, M.M., Mangold, A.J., Anziani, O.S., 2000. Evaluacio’n de una formulacio’n comercial “pour on” con fipronil al 1%
et al., Efficacy to ivermectin
of fipronil (1.0 mg/kg) (0.63 mg/kg). PREVET
against (2014),
G Model PREVET-3571; No. of Pages 6
6
ARTICLE IN PRESS W.D.Z. Lopes et al. / Preventive Veterinary Medicine xxx (2014) xxx–xxx
para el control de Haematobia irritans en vaquillonas Holando naturalmente infestadas. Veterinaria (Argentina) 17, 108–113. Klafke, G.M., Sabatini, G.A., de Albuquerque, T.A., Martins, J.R., Kemp, D.H., Miller, R.J., Schumaker, T.T., 2006. Larval immersion tests with ivermectin in populations of the cattle tick Rhipicephalus (Boophilus) microplus (Acari: Ixodidae) from State of Sao Paulo, Brazil. Vet. Parasitol. 142, 386–390. Lopes, W.D.Z., Teixeira, W.F.P., Matos, L.V.S., Felippelli, G., Cruz, B.C., Maciel, W.G., Buzzulini, C., Fávero, F.C., Soares, V.E., Oliveira, G.P., Costa, A.J., 2013. Effects of macrocyclic lactones on the reproductive parameters of engorged Rhipicephalus (Boophilus) microplus females detached from experimentally infested cattle. Exp. Parasitol. 135, 72–78. Lopes, W.D.Z., Teixeira, W.F.P., Felippelli, G., Cruz, B.C., Maciel, W.G., Soares, V.E., Santos, T.R., Matos, L., Favero, F., Costa, A.J., 2014. Assessing resistance of ivermectin and moxidectin against nematodes in cattle naturally infected using three different methodologies. Res. Vet. Sci. 96, 133–138.
Please cite this article in press as: Lopes, W.D.Z., Rhipicephalus (Boophilus) microplus strains resistant http://dx.doi.org/10.1016/j.prevetmed.2014.04.009
Pereira, J.R., 2009. The efficiency of avermectins (abamectin, doramectin and ivermectin) in the control of Boophilus microplus, in artificially infested bovines kept in field conditions. Vet. Parasitol. 162, 116–119. Roulston, W.J., Wharton, R.H., Nolam, J., Kevr, J.D., 1968. Acaricide tests on the bjarra strain of organophosphorus resistant cattle ticks Boophilus microplus from southern Queensland. Aust. Vet. J. 43, 129–134. Sindicato Nacional da Indústria de Produtos para Saúde Animal – SINDAN, Mercado Veterinário, São Paulo, 2008. Thullner, F., 1997. Impact of pesticide resistance and network for global pesticide resistance management based on a regional structure. WARRMZ 89, 41–47. Wharton, R.H., Utech, K.B.W., 1970. Relation between engorgement and dropping of Boophilus microplus to assessment of tick number in cattle. Aust. Entomol. Soc. 9, 171–182. Zhao, X., Yeh, J.Z., Salgado, V.L., Narahashi, T., 2004. Fipronil is a potent open channel blocker of glutamate-activated chloride channels in cockroach neurons. J. Pharmacol. Exp. Ther. 310, 192–201.
et al., Efficacy to ivermectin
of fipronil (1.0 mg/kg) (0.63 mg/kg). PREVET
against (2014),
![[A 48 kg retroperitoneal giant lipoma].](/assets/img/hold.png)
