Experimental Parasitology 153 (2015) 22–28
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Experimental Parasitology j o u r n a l h o m e p a g e : w w w. e l s e v i e r. c o m / l o c a t e / y e x p r
Full length article
Acaricidal effects of fluazuron (2.5 mg/kg) and a combination of fluazuron (1.6 mg/kg) + ivermectin (0.63 mg/kg), administered at different routes, against Rhipicephalus (Boophilus) microplus parasitizing cattle Lucas Vinicius Costa Gomes a, Welber Daniel Zanetti Lopes a,b,*, Breno Cayeiro Cruz a, Weslen Fabricio Teixeira a, Gustavo Felippelli a, Willian G. Maciel a, Murilo Abud Bichuette a, Maycon A. Ruivo c, Marcos Henrique Alcantara Colli c, Rafael Silveira Carvalho c, Antonio Campanha Martinez c, Vando Edésio Soares a,d, Alvimar José da Costa a a Faculdade de Ciências Agrárias e Veterinárias, UNESP/CPPAR, Via de acesso prof. Paulo Donatto Castellani, s/n CEP:14884-900, Jaboticabal, São PauloBrazil b Universidade Federal de Goiás (UFG), Regional de Jataí, BR-364 Km 192, Parque Industrial, Jataí, GO 75801-615, Brasil cUniversidade Estadual de Maringá (UEM), Campus Regional de Umuarama, Rodov. PR 489, n° 1.400 dUniversidade Camilo Castelo Branco (Unicastelo), Av. Hilário da Silva Passo 950, Descalvado, SP 13690-970, Brasil
• • • •
We compare the acaricide effect of fluazuron administered in different routes. Pour-on fluazuron demonstrated high acaricidal efficacy, against R. microplus. Injectable fluazuron + ivermectin showed efficacy lower/similar to injectable ivermectin. Future studies with the injectable fluazuron + ivermectin should be conducted.
G R A P H I C A L
A B S T R A C T
100 80
Efficacy (%)
H I G H L I G H T S
60 40 20 0 R. (B.) microplus - Artifical infestation
R. (B.) microplus - Natural infestation
Fluazuron (2.5mg/kg), pour-on Fluazuron 1.6mg/kg + ivermectin 0.63mg/kg, injectable Ivermectin 0.63mg/kg, injectable
A R T I C L E
I N F O
Article history: Received 21 May 2014 Received in revised form 9 February 2015 Accepted 11 February 2015 Available online 26 February 2015
A B S T R A C T
The present study aimed to evaluate the acaricidal efficacy of fluazuron (2.5 mg/kg), administered as a pour-on, in comparison to an injectable formulation containing fluazuron (1.6 mg/kg) + ivermectin (0.63 mg/kg), against Rhipicephalus (Boophilus) microplus in naturally and experimentally infested cattle. Two studies were conducted with different tick strains, one with artificial infestations (Stall Test, using
* Corresponding author. Fax +16 3209 2606. E-mail address: [email protected] (W.D.Z. Lopes). http://dx.doi.org/10.1016/j.exppara.2015.02.004 0014-4894/© 2015 Elsevier Inc. All rights reserved.
L.V.C. Gomes et al./Experimental Parasitology 153 (2015) 22–28
Keywords: Engorged females Fluazuron Injectable route Ivermectin Rhipicephalus (Boophilus) microplus
23
leight animals per group) and one with natural infestations (utilizing ten animals per group). In both studies, the animals were randomized, according to average tick counts performed on days −3, −2 and −1, into four groups: T01, negative control (saline solution); T02, pour-on fluazuron (2.5 mg/kg); T03: subcutaneous fluazuron (1.6 mg/kg) + ivermectin (0.63 mg/kg); and T04 subcutaneous ivermectin (0.63 mg/kg). Based on obtained results, and considering the utilized tick strains, it was possible to conclude that the pour-on fluazuron (2.5 mg/kg) formulation demonstrated high acaricidal efficacy, with protection periods ranging from 49 to 77 days against Rhipicephalus (Boophilus) microplus. On the other hand, for the injectable fluazuron (1.6 mg/kg) + ivermectin (0.63 mg/kg) formulation, it was not possible to observe elevated anti-R. (B.) microplus effect on both artificial and experimental infestation studies. Results observed for this combination were similar or inferior to those obtained by subcutaneous ivermectin (0.63 mg/kg). Future studies with this formulation containing fluazuron (1.6 mg/kg) + ivermectin (0.63 mg/kg), regarding pharmacokinetic and/or bioavailability profiles, or even studies analyzing both this active principles separately, are needed, seeking to better understand the effects of such combination against Rhipicephalus (Boophilus) microplus parasitizing cattle. © 2015 Elsevier Inc. All rights reserved.
1. Introduction One of the main obstacles in cattle production is damage caused by Rhipicephalus (Boophilus) microplus (Lopes et al., 2013a). Even though vaccines and biological control are considered possible strategic measures against parasites, these are still in experimental phases and only in the future will be able to compete, on a market level, with conventional therapeutics. Therefore, chemotherapy, in the present, even before the rise of resistant parasite strains, constitutes the most effective tool for tick treatment (Sakamoto et al., 2013). Lately, some chemical control methods include the use of insect growth regulators (Cruz et al., 2014; Maza et al., 2013). This class of antiparasitic compounds (IGRs) can be divided in three categories according to their mode of action: juvenile hormone analogs, chitin synthesis or deposition inhibitors and derivates from the triazine organic compound, which also interferes in pupation and evolution of insects (Chavasse and Yap, 1997; Graf, 1993). Indicated for cattle, we have fluazuron, which is a compound that belongs to the benzoylphenylurea class. It was the first product registered as a growth regulator for controlling Rhipicephalus (Boophilus) microplus (Santos et al., 2010). This active component, used mostly through a topical pour-on route, inhibits chitin synthesis of these parasites, possibly through inhibition of specific enzymes involved on the ecdysis process of ticks (Kemp et al., 1990). Despite the fact that topically administered products are easier to use and present a reduction in costs, without need for needles or syringes; as well as the fact that it does not contribute to pathogen transmission from animal to animal by using the same needles (Silva, 2008), injectable administration route is still more widely used on cattle (Sakamoto et al., 2014). For this reason, a recent injectable (subcutaneous) formulation, based on 8% fluazuron + 3.15% ivermectin, was launched on the Brazilian market (Sindan, 2013). The present study aimed to evaluate acaricidal efficacy of fluazuron (2.5 mg/kg), administered as a pour-on, compared to an injectable formulation containing fluazuron (1.6 mg/kg) + ivermectin (0.63 mg/kg), against R. (B.) microplus parasitizing naturally and artificially infested animals. 2. Material and methods 2.1. Efficacy against artificial infestations of R. (B.) microplus 2.1.1. Location, animals and pre-treatment infestations One experiment was conducted at the Research Center for Animal Health (CPPAR – Centro de Pesquisas em Sanidade Animal) – FCAV/ UNESP/Jaboticabal, state of São Paulo, Brazil. Thirty-two crossbreed bovines, with ages of approximately 7 months, which had not received any antiparasitic treatment for the last 120 days, were selected and identified (numbered ear tags). All
animals were held in individual suspended boxes, appropriate for the conduction of “stall tests”, starting on day 27 before treatment (D-27), for an acclimatization period. After this initial stage, each animal was infested with approximately 5000 R. (B.) microplus larvae (0.25 g of eggs) with ages of 14–28 days, on days −24, −21, −19, −17, −14, −12, −10, −7, −5, −3 and −1, considering day 0 as the treatment date (Ministério da Agricultura e Abastecimento, 1997). This strain of R. (B.) microplus was obtained in a rural property (City of Águas da Prata, São Paulo state, Brazil), and nowadays is kept in CPPAR, using cattle and a BOD incubator. In accordance to owner of such farm, the main compounds used against R. (B.) microplus are macrocyclic lactones, fipronil and fluazuron. 2.1.2. Pre-treatment counts of engorged R. (B.) microplus females detached from bovines and allocation of animals to treatment groups On days −3, −2 and −1, fully engorged R. (B.) microplus females that detached from each animal were counted. All counts were performed daily, in the morning period (between 08:00h and 09:00h a.m.). Animals were randomly allocated on treatment groups according to a randomized complete block design. Block formation was based on arithmetic means of female ticks detached from each animal prior to treatment (days −3, −2 and −1) and individual pen location. In each experiment, animals were divided into eight blocks of four animals each and, inside each block, animals were randomly placed in one of the treatment groups, as represented in Table 1. Blocks were assigned to sets of four nearby pens, and animals within a block were randomly allocated to individual pens within the set. Each animal was an experimental unit. On day 0 (before treatment), all animals were weighed in order to determine the proper dosage of analyzed medications.
Table 1 Experimental design used in study 1 and 2. Treatment
Animals per treatment
Group
Study 1 (artificially infestation) T01 08 Saline solution 0.9% T02 08 Fluazuron 2.5% T03 08 Fluazuron 8.0% + ivermectin 3.15% T04 08 Ivermectin 3.15% Study 2 (naturally infestation) T01 10 Saline solution 0.9% T02 10 Fluazuron 2.5% T03 10 Fluazuron 8.0% + ivermectin 3.15% T04 10 Ivermectin 3.15%
Route
Posology ml/kg
mg/kg
Subcutaneous
1/50
–
Pour-on Subcutaneous
1/10 1/50
Subcutaneous
1/50
2.5 1.6 + 0.63 0.63
Subcutaneous
1/50
–
Pour-on Subcutaneous
1/10 1/50
Subcutaneous
1/50
2.5 1.6 + 0.63 0.63
24
L.V.C. Gomes et al./Experimental Parasitology 153 (2015) 22–28
2.1.3. Counts of engorged R. (B.) microplus females detached from each animal, and post-treatment infestations Engorged female ticks, naturally detached from bovines, were counted daily, starting on day 1 up to the end of each experiment (day 91). During the post-treatment period, all animals were infested with approximately 5000 viable and unfed larvae twice a week (every Tuesday and Thursday of each week), up to the end of the study, as recommended by Holdsworth et al. (2006). Counts of detached engorged female ticks were performed until values of efficacy, obtained by arithmetic means, reached a value less than 70%, with exception of the group that received 1.6 mg/kg fluazuron + 0.63 mg/kg ivermectin.
To evaluate therapeutic and residual efficacies of each formulation, tick counts (female between 4.5 and 8.0 mm long) were performed on days post-treatment (DPT) 3, 7, 14 and every 7 days thereafter until 63 days (Wharton and Utech, 1970). Groups treated with injectable solutions (T01, T03 and T04) were counted first, followed by the group that received topical treatment (T02 – fluazuron 2.5 mg/kg). Disposable gloves were used and changed between tick counts of each animal. Percentages of efficacy against R. (B.) microplus were calculated using arithmetic means, according to a formula proposed by Roulston et al. (1968) and adopted by the Brazilian Ministry of Agriculture, Livestock and Food Supply (Ministério da Agricultura, Pecuária e Abastecimento – MAPA), Ordinance No 48 (Brazil, 1997), as follows:
2.1.4. Efficacy Acaricidal efficacy of each formulation was calculated using arithmetic means of engorged female tick counts, detached from animals of each group, with collected data grouped into 7-day intervals. The following formula was used
⎡ Ta × Cb ⎤ %Efficacy = ⎢1 − × 100 ⎣ Tb × Ca ⎥⎦
⎡ Ta × Cb ⎤ Efficacy percentage = ⎢1 − × 100 ⎣ Tb × Ca ⎥⎦ where Ta = average number of engorged female ticks counted on treated animals post-medication; Tb = average number of engorged female ticks counted on treated animals during the 3 days prior to treatment; Ca = average number of engorged female ticks counted on control animals after the experiment began; Cb = average number of engorged female ticks counted on control animals during the 3 days prior to treatment. 2.2. Efficacy against natural infestations of R. (Boophilus) microplus 2.2.1. Local, division of groups and efficacy calculations A property located in Umuarama City, State of Paraná, Brazil, was used in this study. In accordance to the farm owner, compounds used against R. (B.) microplus are derivates of macrocyclic lactones and fluazuron. Forty crossbreed bovines (Bos taurus) were selected from a herd of approximately 80 animals, all naturally infested with R. (B.) microplus, that had not been treated with ecto or endo-parasiticides during the previous 90 days. Animals were randomly allocated to treatment groups on day 0, according to a randomized complete block design. Block formation was based on arithmetic means of female ticks (4.5–8.0 mm long) in three consecutive days (−3,−2,−1). Animals were divided into ten blocks of four animals each and, inside each block, randomly placed in one of the treatment groups, as represented in Table 1. On day 0, all animals were weighed to determine the appropriate dosage of medication. The number of animals per group used in this study (10 animals per group) was performed in accordance with recommendations described by Brazilian Ministry of Agriculture, Livestock and Food Supply (Ministério da Agricultura, Pecuária e Abastecimento – MAPA), Ordinance No 48 (Brazil, 1997). After treatment administration, the paddock that housed these 40 animals was divided in four. Animals belonging to T01 (control), T03 (fluazuron 1.6 mg/kg + ivermectin 0.63 mg/kg, injectable by subcutaneous route – Contratack injetável® - Clarion Animal Health) and T04 (ivermectin 0.63 mg/kg, injectable by subcutaneous route – Ivomec Gold® – Merial Animal Health) were kept together in three parts of the paddock, while bovines treated with topical fluazuron (T02 – 2.5 mg/kg, pour-on route – Acatak® – Novartis Animal Health Ltda.) were kept on one separate part, so that there would be no contamination between groups due to direct contact among animals. All animals had free access to pasture, water and mineral supplementation throughout the study.
where Ta = mean number of partially engorged ticks counted on treated animals after medication, Tb = mean number of partially engorged ticks counted on treated animals 3 days before treatment, Ca = mean number of partially engorged ticks counted on control animals after the experiment started, Cb = mean number of partially engorged ticks counted on control animals 3 days before treatment. In this study, all procedures using animals complied with the Ethical Principles in Animal Research, adopted by the College of Animal Experimentation (COBEA), being previously approved by the Ethical Committee for Animal Welfare of IPESA, located on the City of Formiga, Minas Gerais State, Brazil. 2.3. Data analysis In the Stall Test, the raw counts of all detached engorged female ticks were grouped into 7-day intervals and log transformed [ln(tick count + 1)]. A generalized linear mixed model for repetitive sampling was applied using SAS (1996), in order to assess fixed effects of treatment, tick counts, interaction between treatment and counts, random block effects and random residual effects. For the natural infestation study, the raw counts of partially engorged ticks on cattle were log transformed using the equation ln (x + 1) prior to statistical analysis. The transformed data were analyzed with a linear mixed model for repeated measures. This model included fixed effects for treatment, study day and their interactions (SAS, 1996). Differences between treatments in both studies (natural and artificially infestation) were evaluated at a significance level of 5% (P ≤ 0.05). 3. Results 3.1. Study 1 (artificial infestation) Regarding efficacy results grouped at 7-day intervals, obtained from animals that were experimentally infested with R. (B.) microplus, it is possible to observe that the formulation containing fluazuron 2.5 mg/kg, administered as a pour-on, presented high acaricidal effects (98.1%) after the 15th day post-treatment (DPT). Such compound reached the maximum value of 100% between 22 and 63 DPT. However, values above 95% were maintained by fluazuron 2.5 mg/kg, pour-on until the 77th DPT, when, in sequence, the efficacy of such compound decreased to 87.5% and 71.5%, between days 78–84 and 85–91 post-treatment, respectively (Table 2). When we analyze the injectable combination containing fluazuron (1.6 mg/kg) + ivermectin (0.63 mg/kg), acaricidal values of such compound against R. (B.) microplus were considered low when compared to indexes obtained by the previous formulation.
L.V.C. Gomes et al./Experimental Parasitology 153 (2015) 22–28
25
Table 2 Average number of engorged Rhipicephalus (Boophilus) microplus females naturally detached from control and treated cattle and percent efficacy (arithmetic means), Study 1. Day of study
Zero 1–7 8–14 15–21 22–28 29–35 36–42 43–49 50–56 57–63 64–71 72–77 78–84 85–91 a =
Experimental groups/Mean value of engorged females detached of cattle T01: Control (saline solution)
T02: 2.5 mg/kg fluazuron
47.67 63.29 70.89 74.75 78.36 80.25 80.89 81.29 81.13 85.16 91.88 100.20 99.57 94.36
49.21 67.07 41.80 1.48 0.00 0.02 0.00 0.00 0.00 0.00 1.05 4.98 12.82 27.73
a
Efficacy (%)
T03: 1.6 mg/kg fluazuron + 0.63 mg/kg ivermectina
T04: 0.63 mg/kg
49.88 52.80 35.48 40.88 39.80 24.21 19.63 23.64 30.89 43.91 56.96 58.98 65.30 75.45
49.13 47.13 19.55 10.75 8.02 8.16 12.41 15.50 18.21 36.69 – – – –
ivermectina
T02
T03
T04
–
– 20.3 52.2 47.7 51.5 71.2 76.8 72.2 63.6 50.7 40.7 43.7 37.3 23.6
– 27.8 73.2 86.0 90.1 90.1 85.1 81.5 78.2 58.2 – – – –
0.0 42.9 98.1 100.0 100.0 100.0 100.0 100.0 100.0 98.9 95.2 87.5 71.5
Commercial formulation purchased in the local market.
Table 3 Results of multiple comparisons of the engorged Rhipicephalus (Boophilus) microplus females naturally detached from control and treated cattle, Study 1. Day of study
Experimental groups/mean [ln(x+1)]/n]a and standard deviation Control (saline solution)
Zero 1–7 8–14 15–21 22–28 29–35 36–42 43–49 50–56 57–63 64 to 70 71 to 77 78 to 84 85 to 91 a b c d
1.6736 1.7975 1.8463 1.8620 1.8856 1.8985 1.9047 1.9121 1.9095 1.9317 1.9609 1.9951 1.9917 1.9702
± ± ± ± ± ± ± ± ± ± ± ± ± ±
0.1141 0.0803 0.0846 0.1298 0.1078 0.0952 0.0803 0.0323 0.0543 0.0469 0.0668 0.0855 0.0904 0.0882
A A A A A A A A A A A A A A
1.7729 ± 0.1072 1.8081 ± 0.1467 1.5856 ± 0.2075 0.2542 ± 0.2227 0.0000 ± 0.0000 0.0000 ± 0.0000 0.0000 ± 0.0000 0.0000 ± 0.0000 0.0000 ± 0.0000 0.0000 ± 0.0000 0.1725 ± 0.2800 0.6893 ± 0.2363 1.0491 ± 0.2481 1.3708 ± 0.950
Variance of analysis
1.6 mg/kg fluazuron + 0.63 mg/kg ivermectind
2.5 mg/kg fluazurond A A B D D D D D C C C C C B
1.7499 1.7144 1.5286 1.5243 1.4557 1.2631 1.2481 1.3312 1.4035 1.5477 1.6609 1.6607 1.7253 1.8062
± ± ± ± ± ± ± ± ± ± ± ± ± ±
0.1199 0.1072 0.1721 0.3455 0.4209 0.3377 0.2481 0.2458 0.3403 0.3359 0.3268 0.3563 0.3137 0.2855
A A B B B B B B B B B B B B
0.63 mg/kg ivermectind 1.7499 ± 0.1199 1.6666 ± 0.1030 1.2658 ± 0.2056 0.9669 ± 0.3030 0.9131 ± 0.1479 0.9288 ± 0.1168 1.0487 ± 0.2394 1.0865 ± 0.3912 1.2010 ± 0.2831 1.5228 ± 0.2279 – – – –
A B C C C C C C B B
Value of Fb
Prob. < Fc
0.39 0.95 11.66 101.67 135.96 128.84 128.35 131.75 134.66 150.14 176.81 88.68 45.51 18.49
0.7627 0.4186
Contents lists available at ScienceDirect
Experimental Parasitology j o u r n a l h o m e p a g e : w w w. e l s e v i e r. c o m / l o c a t e / y e x p r
Full length article
Acaricidal effects of fluazuron (2.5 mg/kg) and a combination of fluazuron (1.6 mg/kg) + ivermectin (0.63 mg/kg), administered at different routes, against Rhipicephalus (Boophilus) microplus parasitizing cattle Lucas Vinicius Costa Gomes a, Welber Daniel Zanetti Lopes a,b,*, Breno Cayeiro Cruz a, Weslen Fabricio Teixeira a, Gustavo Felippelli a, Willian G. Maciel a, Murilo Abud Bichuette a, Maycon A. Ruivo c, Marcos Henrique Alcantara Colli c, Rafael Silveira Carvalho c, Antonio Campanha Martinez c, Vando Edésio Soares a,d, Alvimar José da Costa a a Faculdade de Ciências Agrárias e Veterinárias, UNESP/CPPAR, Via de acesso prof. Paulo Donatto Castellani, s/n CEP:14884-900, Jaboticabal, São PauloBrazil b Universidade Federal de Goiás (UFG), Regional de Jataí, BR-364 Km 192, Parque Industrial, Jataí, GO 75801-615, Brasil cUniversidade Estadual de Maringá (UEM), Campus Regional de Umuarama, Rodov. PR 489, n° 1.400 dUniversidade Camilo Castelo Branco (Unicastelo), Av. Hilário da Silva Passo 950, Descalvado, SP 13690-970, Brasil
• • • •
We compare the acaricide effect of fluazuron administered in different routes. Pour-on fluazuron demonstrated high acaricidal efficacy, against R. microplus. Injectable fluazuron + ivermectin showed efficacy lower/similar to injectable ivermectin. Future studies with the injectable fluazuron + ivermectin should be conducted.
G R A P H I C A L
A B S T R A C T
100 80
Efficacy (%)
H I G H L I G H T S
60 40 20 0 R. (B.) microplus - Artifical infestation
R. (B.) microplus - Natural infestation
Fluazuron (2.5mg/kg), pour-on Fluazuron 1.6mg/kg + ivermectin 0.63mg/kg, injectable Ivermectin 0.63mg/kg, injectable
A R T I C L E
I N F O
Article history: Received 21 May 2014 Received in revised form 9 February 2015 Accepted 11 February 2015 Available online 26 February 2015
A B S T R A C T
The present study aimed to evaluate the acaricidal efficacy of fluazuron (2.5 mg/kg), administered as a pour-on, in comparison to an injectable formulation containing fluazuron (1.6 mg/kg) + ivermectin (0.63 mg/kg), against Rhipicephalus (Boophilus) microplus in naturally and experimentally infested cattle. Two studies were conducted with different tick strains, one with artificial infestations (Stall Test, using
* Corresponding author. Fax +16 3209 2606. E-mail address: [email protected] (W.D.Z. Lopes). http://dx.doi.org/10.1016/j.exppara.2015.02.004 0014-4894/© 2015 Elsevier Inc. All rights reserved.
L.V.C. Gomes et al./Experimental Parasitology 153 (2015) 22–28
Keywords: Engorged females Fluazuron Injectable route Ivermectin Rhipicephalus (Boophilus) microplus
23
leight animals per group) and one with natural infestations (utilizing ten animals per group). In both studies, the animals were randomized, according to average tick counts performed on days −3, −2 and −1, into four groups: T01, negative control (saline solution); T02, pour-on fluazuron (2.5 mg/kg); T03: subcutaneous fluazuron (1.6 mg/kg) + ivermectin (0.63 mg/kg); and T04 subcutaneous ivermectin (0.63 mg/kg). Based on obtained results, and considering the utilized tick strains, it was possible to conclude that the pour-on fluazuron (2.5 mg/kg) formulation demonstrated high acaricidal efficacy, with protection periods ranging from 49 to 77 days against Rhipicephalus (Boophilus) microplus. On the other hand, for the injectable fluazuron (1.6 mg/kg) + ivermectin (0.63 mg/kg) formulation, it was not possible to observe elevated anti-R. (B.) microplus effect on both artificial and experimental infestation studies. Results observed for this combination were similar or inferior to those obtained by subcutaneous ivermectin (0.63 mg/kg). Future studies with this formulation containing fluazuron (1.6 mg/kg) + ivermectin (0.63 mg/kg), regarding pharmacokinetic and/or bioavailability profiles, or even studies analyzing both this active principles separately, are needed, seeking to better understand the effects of such combination against Rhipicephalus (Boophilus) microplus parasitizing cattle. © 2015 Elsevier Inc. All rights reserved.
1. Introduction One of the main obstacles in cattle production is damage caused by Rhipicephalus (Boophilus) microplus (Lopes et al., 2013a). Even though vaccines and biological control are considered possible strategic measures against parasites, these are still in experimental phases and only in the future will be able to compete, on a market level, with conventional therapeutics. Therefore, chemotherapy, in the present, even before the rise of resistant parasite strains, constitutes the most effective tool for tick treatment (Sakamoto et al., 2013). Lately, some chemical control methods include the use of insect growth regulators (Cruz et al., 2014; Maza et al., 2013). This class of antiparasitic compounds (IGRs) can be divided in three categories according to their mode of action: juvenile hormone analogs, chitin synthesis or deposition inhibitors and derivates from the triazine organic compound, which also interferes in pupation and evolution of insects (Chavasse and Yap, 1997; Graf, 1993). Indicated for cattle, we have fluazuron, which is a compound that belongs to the benzoylphenylurea class. It was the first product registered as a growth regulator for controlling Rhipicephalus (Boophilus) microplus (Santos et al., 2010). This active component, used mostly through a topical pour-on route, inhibits chitin synthesis of these parasites, possibly through inhibition of specific enzymes involved on the ecdysis process of ticks (Kemp et al., 1990). Despite the fact that topically administered products are easier to use and present a reduction in costs, without need for needles or syringes; as well as the fact that it does not contribute to pathogen transmission from animal to animal by using the same needles (Silva, 2008), injectable administration route is still more widely used on cattle (Sakamoto et al., 2014). For this reason, a recent injectable (subcutaneous) formulation, based on 8% fluazuron + 3.15% ivermectin, was launched on the Brazilian market (Sindan, 2013). The present study aimed to evaluate acaricidal efficacy of fluazuron (2.5 mg/kg), administered as a pour-on, compared to an injectable formulation containing fluazuron (1.6 mg/kg) + ivermectin (0.63 mg/kg), against R. (B.) microplus parasitizing naturally and artificially infested animals. 2. Material and methods 2.1. Efficacy against artificial infestations of R. (B.) microplus 2.1.1. Location, animals and pre-treatment infestations One experiment was conducted at the Research Center for Animal Health (CPPAR – Centro de Pesquisas em Sanidade Animal) – FCAV/ UNESP/Jaboticabal, state of São Paulo, Brazil. Thirty-two crossbreed bovines, with ages of approximately 7 months, which had not received any antiparasitic treatment for the last 120 days, were selected and identified (numbered ear tags). All
animals were held in individual suspended boxes, appropriate for the conduction of “stall tests”, starting on day 27 before treatment (D-27), for an acclimatization period. After this initial stage, each animal was infested with approximately 5000 R. (B.) microplus larvae (0.25 g of eggs) with ages of 14–28 days, on days −24, −21, −19, −17, −14, −12, −10, −7, −5, −3 and −1, considering day 0 as the treatment date (Ministério da Agricultura e Abastecimento, 1997). This strain of R. (B.) microplus was obtained in a rural property (City of Águas da Prata, São Paulo state, Brazil), and nowadays is kept in CPPAR, using cattle and a BOD incubator. In accordance to owner of such farm, the main compounds used against R. (B.) microplus are macrocyclic lactones, fipronil and fluazuron. 2.1.2. Pre-treatment counts of engorged R. (B.) microplus females detached from bovines and allocation of animals to treatment groups On days −3, −2 and −1, fully engorged R. (B.) microplus females that detached from each animal were counted. All counts were performed daily, in the morning period (between 08:00h and 09:00h a.m.). Animals were randomly allocated on treatment groups according to a randomized complete block design. Block formation was based on arithmetic means of female ticks detached from each animal prior to treatment (days −3, −2 and −1) and individual pen location. In each experiment, animals were divided into eight blocks of four animals each and, inside each block, animals were randomly placed in one of the treatment groups, as represented in Table 1. Blocks were assigned to sets of four nearby pens, and animals within a block were randomly allocated to individual pens within the set. Each animal was an experimental unit. On day 0 (before treatment), all animals were weighed in order to determine the proper dosage of analyzed medications.
Table 1 Experimental design used in study 1 and 2. Treatment
Animals per treatment
Group
Study 1 (artificially infestation) T01 08 Saline solution 0.9% T02 08 Fluazuron 2.5% T03 08 Fluazuron 8.0% + ivermectin 3.15% T04 08 Ivermectin 3.15% Study 2 (naturally infestation) T01 10 Saline solution 0.9% T02 10 Fluazuron 2.5% T03 10 Fluazuron 8.0% + ivermectin 3.15% T04 10 Ivermectin 3.15%
Route
Posology ml/kg
mg/kg
Subcutaneous
1/50
–
Pour-on Subcutaneous
1/10 1/50
Subcutaneous
1/50
2.5 1.6 + 0.63 0.63
Subcutaneous
1/50
–
Pour-on Subcutaneous
1/10 1/50
Subcutaneous
1/50
2.5 1.6 + 0.63 0.63
24
L.V.C. Gomes et al./Experimental Parasitology 153 (2015) 22–28
2.1.3. Counts of engorged R. (B.) microplus females detached from each animal, and post-treatment infestations Engorged female ticks, naturally detached from bovines, were counted daily, starting on day 1 up to the end of each experiment (day 91). During the post-treatment period, all animals were infested with approximately 5000 viable and unfed larvae twice a week (every Tuesday and Thursday of each week), up to the end of the study, as recommended by Holdsworth et al. (2006). Counts of detached engorged female ticks were performed until values of efficacy, obtained by arithmetic means, reached a value less than 70%, with exception of the group that received 1.6 mg/kg fluazuron + 0.63 mg/kg ivermectin.
To evaluate therapeutic and residual efficacies of each formulation, tick counts (female between 4.5 and 8.0 mm long) were performed on days post-treatment (DPT) 3, 7, 14 and every 7 days thereafter until 63 days (Wharton and Utech, 1970). Groups treated with injectable solutions (T01, T03 and T04) were counted first, followed by the group that received topical treatment (T02 – fluazuron 2.5 mg/kg). Disposable gloves were used and changed between tick counts of each animal. Percentages of efficacy against R. (B.) microplus were calculated using arithmetic means, according to a formula proposed by Roulston et al. (1968) and adopted by the Brazilian Ministry of Agriculture, Livestock and Food Supply (Ministério da Agricultura, Pecuária e Abastecimento – MAPA), Ordinance No 48 (Brazil, 1997), as follows:
2.1.4. Efficacy Acaricidal efficacy of each formulation was calculated using arithmetic means of engorged female tick counts, detached from animals of each group, with collected data grouped into 7-day intervals. The following formula was used
⎡ Ta × Cb ⎤ %Efficacy = ⎢1 − × 100 ⎣ Tb × Ca ⎥⎦
⎡ Ta × Cb ⎤ Efficacy percentage = ⎢1 − × 100 ⎣ Tb × Ca ⎥⎦ where Ta = average number of engorged female ticks counted on treated animals post-medication; Tb = average number of engorged female ticks counted on treated animals during the 3 days prior to treatment; Ca = average number of engorged female ticks counted on control animals after the experiment began; Cb = average number of engorged female ticks counted on control animals during the 3 days prior to treatment. 2.2. Efficacy against natural infestations of R. (Boophilus) microplus 2.2.1. Local, division of groups and efficacy calculations A property located in Umuarama City, State of Paraná, Brazil, was used in this study. In accordance to the farm owner, compounds used against R. (B.) microplus are derivates of macrocyclic lactones and fluazuron. Forty crossbreed bovines (Bos taurus) were selected from a herd of approximately 80 animals, all naturally infested with R. (B.) microplus, that had not been treated with ecto or endo-parasiticides during the previous 90 days. Animals were randomly allocated to treatment groups on day 0, according to a randomized complete block design. Block formation was based on arithmetic means of female ticks (4.5–8.0 mm long) in three consecutive days (−3,−2,−1). Animals were divided into ten blocks of four animals each and, inside each block, randomly placed in one of the treatment groups, as represented in Table 1. On day 0, all animals were weighed to determine the appropriate dosage of medication. The number of animals per group used in this study (10 animals per group) was performed in accordance with recommendations described by Brazilian Ministry of Agriculture, Livestock and Food Supply (Ministério da Agricultura, Pecuária e Abastecimento – MAPA), Ordinance No 48 (Brazil, 1997). After treatment administration, the paddock that housed these 40 animals was divided in four. Animals belonging to T01 (control), T03 (fluazuron 1.6 mg/kg + ivermectin 0.63 mg/kg, injectable by subcutaneous route – Contratack injetável® - Clarion Animal Health) and T04 (ivermectin 0.63 mg/kg, injectable by subcutaneous route – Ivomec Gold® – Merial Animal Health) were kept together in three parts of the paddock, while bovines treated with topical fluazuron (T02 – 2.5 mg/kg, pour-on route – Acatak® – Novartis Animal Health Ltda.) were kept on one separate part, so that there would be no contamination between groups due to direct contact among animals. All animals had free access to pasture, water and mineral supplementation throughout the study.
where Ta = mean number of partially engorged ticks counted on treated animals after medication, Tb = mean number of partially engorged ticks counted on treated animals 3 days before treatment, Ca = mean number of partially engorged ticks counted on control animals after the experiment started, Cb = mean number of partially engorged ticks counted on control animals 3 days before treatment. In this study, all procedures using animals complied with the Ethical Principles in Animal Research, adopted by the College of Animal Experimentation (COBEA), being previously approved by the Ethical Committee for Animal Welfare of IPESA, located on the City of Formiga, Minas Gerais State, Brazil. 2.3. Data analysis In the Stall Test, the raw counts of all detached engorged female ticks were grouped into 7-day intervals and log transformed [ln(tick count + 1)]. A generalized linear mixed model for repetitive sampling was applied using SAS (1996), in order to assess fixed effects of treatment, tick counts, interaction between treatment and counts, random block effects and random residual effects. For the natural infestation study, the raw counts of partially engorged ticks on cattle were log transformed using the equation ln (x + 1) prior to statistical analysis. The transformed data were analyzed with a linear mixed model for repeated measures. This model included fixed effects for treatment, study day and their interactions (SAS, 1996). Differences between treatments in both studies (natural and artificially infestation) were evaluated at a significance level of 5% (P ≤ 0.05). 3. Results 3.1. Study 1 (artificial infestation) Regarding efficacy results grouped at 7-day intervals, obtained from animals that were experimentally infested with R. (B.) microplus, it is possible to observe that the formulation containing fluazuron 2.5 mg/kg, administered as a pour-on, presented high acaricidal effects (98.1%) after the 15th day post-treatment (DPT). Such compound reached the maximum value of 100% between 22 and 63 DPT. However, values above 95% were maintained by fluazuron 2.5 mg/kg, pour-on until the 77th DPT, when, in sequence, the efficacy of such compound decreased to 87.5% and 71.5%, between days 78–84 and 85–91 post-treatment, respectively (Table 2). When we analyze the injectable combination containing fluazuron (1.6 mg/kg) + ivermectin (0.63 mg/kg), acaricidal values of such compound against R. (B.) microplus were considered low when compared to indexes obtained by the previous formulation.
L.V.C. Gomes et al./Experimental Parasitology 153 (2015) 22–28
25
Table 2 Average number of engorged Rhipicephalus (Boophilus) microplus females naturally detached from control and treated cattle and percent efficacy (arithmetic means), Study 1. Day of study
Zero 1–7 8–14 15–21 22–28 29–35 36–42 43–49 50–56 57–63 64–71 72–77 78–84 85–91 a =
Experimental groups/Mean value of engorged females detached of cattle T01: Control (saline solution)
T02: 2.5 mg/kg fluazuron
47.67 63.29 70.89 74.75 78.36 80.25 80.89 81.29 81.13 85.16 91.88 100.20 99.57 94.36
49.21 67.07 41.80 1.48 0.00 0.02 0.00 0.00 0.00 0.00 1.05 4.98 12.82 27.73
a
Efficacy (%)
T03: 1.6 mg/kg fluazuron + 0.63 mg/kg ivermectina
T04: 0.63 mg/kg
49.88 52.80 35.48 40.88 39.80 24.21 19.63 23.64 30.89 43.91 56.96 58.98 65.30 75.45
49.13 47.13 19.55 10.75 8.02 8.16 12.41 15.50 18.21 36.69 – – – –
ivermectina
T02
T03
T04
–
– 20.3 52.2 47.7 51.5 71.2 76.8 72.2 63.6 50.7 40.7 43.7 37.3 23.6
– 27.8 73.2 86.0 90.1 90.1 85.1 81.5 78.2 58.2 – – – –
0.0 42.9 98.1 100.0 100.0 100.0 100.0 100.0 100.0 98.9 95.2 87.5 71.5
Commercial formulation purchased in the local market.
Table 3 Results of multiple comparisons of the engorged Rhipicephalus (Boophilus) microplus females naturally detached from control and treated cattle, Study 1. Day of study
Experimental groups/mean [ln(x+1)]/n]a and standard deviation Control (saline solution)
Zero 1–7 8–14 15–21 22–28 29–35 36–42 43–49 50–56 57–63 64 to 70 71 to 77 78 to 84 85 to 91 a b c d
1.6736 1.7975 1.8463 1.8620 1.8856 1.8985 1.9047 1.9121 1.9095 1.9317 1.9609 1.9951 1.9917 1.9702
± ± ± ± ± ± ± ± ± ± ± ± ± ±
0.1141 0.0803 0.0846 0.1298 0.1078 0.0952 0.0803 0.0323 0.0543 0.0469 0.0668 0.0855 0.0904 0.0882
A A A A A A A A A A A A A A
1.7729 ± 0.1072 1.8081 ± 0.1467 1.5856 ± 0.2075 0.2542 ± 0.2227 0.0000 ± 0.0000 0.0000 ± 0.0000 0.0000 ± 0.0000 0.0000 ± 0.0000 0.0000 ± 0.0000 0.0000 ± 0.0000 0.1725 ± 0.2800 0.6893 ± 0.2363 1.0491 ± 0.2481 1.3708 ± 0.950
Variance of analysis
1.6 mg/kg fluazuron + 0.63 mg/kg ivermectind
2.5 mg/kg fluazurond A A B D D D D D C C C C C B
1.7499 1.7144 1.5286 1.5243 1.4557 1.2631 1.2481 1.3312 1.4035 1.5477 1.6609 1.6607 1.7253 1.8062
± ± ± ± ± ± ± ± ± ± ± ± ± ±
0.1199 0.1072 0.1721 0.3455 0.4209 0.3377 0.2481 0.2458 0.3403 0.3359 0.3268 0.3563 0.3137 0.2855
A A B B B B B B B B B B B B
0.63 mg/kg ivermectind 1.7499 ± 0.1199 1.6666 ± 0.1030 1.2658 ± 0.2056 0.9669 ± 0.3030 0.9131 ± 0.1479 0.9288 ± 0.1168 1.0487 ± 0.2394 1.0865 ± 0.3912 1.2010 ± 0.2831 1.5228 ± 0.2279 – – – –
A B C C C C C C B B
Value of Fb
Prob. < Fc
0.39 0.95 11.66 101.67 135.96 128.84 128.35 131.75 134.66 150.14 176.81 88.68 45.51 18.49
0.7627 0.4186
