- Email: [email protected]
Dermatobia hominis: Potencial risk of resistance to macrocyclic lactones
Accepted Manuscript Title: Dermatobia hominis: potencial risk of resistance to macrocyclic lactones Author: Jos´e Henrique das Neves Nadino Carvalho Alessandro F.T. Amarante PII: DOI: Reference:
S0304-4017(15)00324-6 http://dx.doi.org/doi:10.1016/j.vetpar.2015.06.029 VETPAR 7682
To appear in:
Veterinary Parasitology
Received date: Revised date: Accepted date:
27-4-2015 22-6-2015 23-6-2015
Please cite this article as: Neves, Jos´e Henrique das, Carvalho, Nadino, Amarante, Alessandro F.T., Dermatobia hominis: potencial risk of resistance to macrocyclic lactones.Veterinary Parasitology http://dx.doi.org/10.1016/j.vetpar.2015.06.029 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Dermatobia hominis: potencial risk of resistance to macrocyclic lactones
José Henrique das Nevesa*, Nadino Carvalhoa, Alessandro F. T. Amarantea
a
UNESP – Universidade Estadual Paulista Júlio de Mesquita Filho, Departamento de
Parasitologia, IBB, CEP 18618-970, Botucatu, SP, Brazil. *Corresponding author: [email protected]
Highlights
•
First report of Dermatobia hominis resistance to ivermectin.
•
Several larvae found to have survived the ivermectin treatment.
•
The effectiveness of moxidectin to control D. hominis was low.
Abstract
Dermatobia hominis is an ectoparasite that infests various species of mammals, including cattle, impairing the quality of cowhides and leather. After observing natural infestation with D. hominis larvae in cattle on two farms in the state of São Paulo, Brazil, we evaluated the efficacy of two macrocyclic lactones, ivermectin and moxidectin, against this parasite. The drugs were administered to 10 animals in each group, following the manufacturer’s instructions. The groups were: Group 1 – treated with ivermectin (0.2 mg/kg of body weight (BW)); Group 2 – treated with moxidectin (0.2 mg/kg BW); and Group 3 – control (untreated). On the farm in Pardinho, a total of 12 and 16 live larvae were found in 6 and in 8 animals 10 days after the treatment with ivermectin and moxidectin, respectively, while in the control group 4 bovines had a total of 7 live larvae. On the farm in Anhembi, 2, 4 and 6 live larvae were extracted from ivermectin, moxidectin and control groups, respectively, after the treatment. This
is the first report of the presence of live D. hominis larvae after the treatment of cattle with ivermectin and moxidectin in Brazil. Keywords: Drug resistance, ivermectin, macrocyclic lactones
1. Introduction Dermatobia hominis is an ectoparasite that affects several mammal species, such as pigs, goats, dogs, cats, humans (Silva-Junior et al., 1998), sheep (Madeira et al., 2000), rabbits (Verocai et al., 2009), cattle (Silva et al., 2010), and sometimes horses (Daemon and Prata, 1997). This ectoparasite, which also parasitizes humans (Hu et al., 2013), occurs from the southern North America to southern Argentina (Hill and Connelly, 2011). During flight, D. hominis adult flies capture phoretic flies, upon whose ventral abdominal region they deposit 4 to 52 eggs (Gomes et al., 2002; Maia and Gomes, 1988). There are reports of 46 different vectors, including mosquitoes, flies and ticks (Guimarães et al., 1983). After 4 to 6 days of incubation, a first instar larvae is formed inside the egg (Barbosa et al., 2002). The larvae actively penetrate the host’s skin and begin their development in the subcutaneous tissue (Leite et al., 1999). In experimental infestations of D. hominis in cattle, it was observed that, in the initial days after infestation, the holes in the skin that enable the larvae to breath are practically imperceptible, but 7 days after infestation, nodules become visible in the subcutaneous tissue. At around day 14 post-infestation, the larvae molt into the second larval stage (L2) and nodules are approximately 1 cm in size. The nodules progressively increase in size, reaching 2 to 3 cm in diameter by 30 days post-infestation. At this point, exudate is eliminated through the hole through which the L3 larvae breathe.
Between day 33 and 42 post-infestation, L3 larvae complete their development and begin to leave the host (Barbosa et al., 2002). Although some studies report losses resulting from combined (simultaneous) parasitism by the gastrointestinal nematodes, Haematobia irritans, Rhipicephalus microplus and D. hominis (Bianchin et al., 2007; Netto et al., 2006), the major financial loss caused by D. hominis infestations is the decrease in leather quality due to perforations (Gomes, 2002; Marques et al., 2000). Today, leather is the second largest export product of the Brazilian cattle industry, generating annual revenues of over 2.48 billion dollars (ANUALPEC, 2014). However, about 40% of the leather produced in Brazil has low commercial value due to injuries caused by ectoparasite infestations, and 89% of the leather produced in the states of Mato Grosso, Minas Gerais, Rio de Janeiro and Sao Paulo States has perforations caused by D. hominis (Gomes, 2002). In Brazil, endoparasite and ectoparasite infestations in cattle are controlled mainly with drugs, especially with the macrocyclic lactone class of endectocides. However, there have been frequent reports of anthelmintic resistance (Neves et al., 2014), and also reports of R. microplus (Reck et al., 2014) and Cochliomyia hominivorax (Lopes et al., 2013) resistance to treatment with macrocyclic lactones. This paper describes, for the first time, the presence of live D. hominis larvae after the treatment of cattle with ivermectin and moxidectin in Brazil.
2. Materials and methods This work was developed in accordance with the ethical principles of animal experimentation and was approved by the Ethics Committee on Animal Use of the School of Veterinary Medicine and Animal Science of UNESP at Botucatu (Protocol 44/2012/CEUA-FMVZ).
2.1. Cattle Herds and Management In February and March 2013, during an experiment conducted to evaluate anthelmintic resistance in cattle (Neves et al., 2014), we observed, by visual inspection, that most of the animals of two farms were presenting the typical lesions on skin caused by D. hominis infestation. For this reason, we decided to also evaluate the efficacy of macrocyclic lactones against D. hominis larvae in the cattle on those farms; one located in the municipality of Pardinho and one in the municipality Anhembi, both in the state of São Paulo, Brazil. The animals of the farm in Pardinho were Holstein heifers, with a mean age (± standard deviation) of 12.5±3.92 months. The animals of the farm in Anhembi were Nellore male calves, with a mean age of 8.26±0.66 months.
2.2. Experimental procedure The animals were weighed and separated into groups in order to evaluate the efficacy of anthelmintics (Neves et al., 2014). The drugs were administered to 10 animals in each group, given by subcutaneous injection following the manufacturer’s instructions. The groups were: Group 1 – treated with ivermectin (0.2 mg/kg of body weight (BW), Ivomec®, Merial, Brazil); Group 2 – treated with moxidectin (0.2 mg/kg BW, Cydectin®, Fort Dodge, Brazil); Group 3 – control (untreated).
2.3. Physical examination of the animals Ten days after the drugs were administered, the animals were immobilized for a visual inspection and palpation of all the nodules over their entire bodies. The larvae were extracted by manual compression of the nodules, and checked to determine if they were moving, i.e., live, as recommended by the World Association for the Advancement
of Veterinary Parasitology (WAAVP) (Holdsworth et al., 2006). These procedures were photographed and filmed. The manual compression of the nodules was not performed on the day of the treatment to avoid damaging or killing the larvae, which could cause confusion with the efficacy of the drug treatment.
2.4. Statistical analysis The efficacy was calculated according to the following formula: 100 x [(CT)/C], to determine the geometric mean parasite density of the treated (T) and control (C) animals. For a therapeutic claim a reduction of larvae counts should be at least 90%, calculated by Abbott’s formula (Abbott, 1925) to compare treated and control animal groups (Holdsworth et al., 2006).
3. Results 3.1. History of use and criteria for the choice of anthelmintics The manager of the farm in Pardinho reported that the body weight of the animals was recorded prior to the administration of endectocides in order to determine the correct dosage of the drugs, according to manufacturer’s recommendation. Ivermectin, abamectin or doramectin were administered six times per year. The manager also reported that he had administered a combination of pour-on abamectin + fluazuron (3.0 mg/kg fluazuron + 0.5 mg/kg Abamectin, Fluatac Duo®, Ouro Fino, Brazil) about 60 days prior to the experiment. The owner of the farm in Anhembi reported that the calculation of the dosage of endectocides was based on a visual estimate of the animals’ body weight. Ivermectin or moxidectin were administered 3 times a year. In addition, Fentione (15 mg/kg;
Tiguvon® 15 Spot-On®, Bayer Animal Health, Brazil) was applied to control D. hominis whenever the parasite was detected visually.
3.2 Capture of a Dipterous Vector During the collection of data in the farm in Pardinho, large numbers of flies were observed in the environment and on the animals. In addition a fly bearing eggs of D. hominis was captured and placed in a jar with a lid.
The specimen, which was
identified as Musca domestica based on its morphological wing and antenna structures, was carrying 24 eggs of D. hominis attached to the lateral-ventral region of its abdomen (Figure 1).
3.3 Efficacy against Dermatobia hominis The efficacy of moxidectin and ivermectin on the farm in Pardinho was zero (Table 1), i.e., the count of viable larvae (Figure 2) recovered from the groups treated with moxidectin or ivermectin was higher than in the control group. At this farm, 3 bovines showed one live larvae, while other 3 animals presented two, three or four live larvae, after treatment with ivermectin (Table 1). Of the 10 animals treated with moxidectin, eight had live larvae: five animals with one larvae; two animals with two larvae; and one bovine with seven larvae. In the control group 4 bovines had a total of 7 live larvae (Table 1). On the farm in Anhembi, the degree of infestation was lower: 2, 4 and 6 live larvae were extracted from cattle of the ivermectin, moxidectin and control groups, respectively, corresponding to an efficacy of 67% and 33% (Table 1).
4. Discussion
Despite the relatively small number of larvae parasitizing the animals, several larvae were found to have survived the treatment. The efficacy of ivermectin in cattle infested with D. hominis is considered very high, with reports of 100% efficacy 7 to 10 days post treatment (Inforzato et al., 2008; Oliveira-Sequeira et al., 2014). Therefore, this is the first study in which live D. hominis larvae was found after treatment of cattle with ivermectin. The effectiveness of moxidectin to control D. hominis was also low. However, this anthelmintic does not have a registered statement of effectiveness against D. hominis, although it does against Hypoderma sp., which is a parasite not found in Brazil. The resistance of D. hominis to ivermectin is a very important issue because this drug is used extensively on Brazilian cattle (Neves et al., 2014). It is assumed that, with the continued use of the product, resistance to it will spread across the country, with negative consequences to the leather industry. The diptera (M. domestica) that was carrying the eggs of D. hominis has already been described as a vector in the state of Minas Gerais (Rodríguez and Leite, 1997). However, this is the first time M. domestica was found bearing eggs of D. hominis in the state of São Paulo. It should be noted that M. domestica is a synanthropic fly that occurs abundantly throughout the state of São Paulo and, according to Monteiro and Prado (2000), it can be found all year round.
Conclusion This is the first report of live D. hominis larvae found after the treatment of cattle with macrocyclic lactone (ivermectin), indicating development of resistance in such species.
Acknowledgements José Henrique das Neves and Nadino Carvalho gratefully acknowledge the financial support of CAPES (Brazil’s Federal Agency for the Support and Improvement of Higher Education) and Alessandro F. T. Amarante acknowledges the support of CNPq (Brazil’s National Council for Scientific and Technological Development).
References
Abbott, W.S., 1925. A method of computing the effectiveness of an insecticide. J. Econ. Entomol. 18, 265–267. ANUALPEC, 2013. Anuário da Pecuária Brasileira, São Paulo: FNP. São Paulo. Barbosa, C.G., Sanavria, A., Barbosa, M.D.P.R.C., 2002. Fase parasitária e alterações clínicas em bovinos infestados experimentalmente com larvas de Dermatobia hominis (Diptera: Cuterebridae). Parasitol. Latinoam. 57, 15–20. Bianchin, I., Catto, J.B., Kichel, A.N., Torres, R.A.A., Honer, M.R., 2007. The effect of the control of endo- and ectoparasites on weight gains in crossbred cattle (Bos taurus taurus×Bos taurus indicus) in the central region of Brazil. Trop. Anim. Health Prod. 39, 287–296. Daemon, E., Prata, M.C.A., 1997. Miíase conjuntival por larvas de Dermatobia hominis (Linnaeus Jr., 1781) (Diptera, Cuterebridae) em equino: Descrição de um caso. Rev. Bras. Med. Veterinária 19, 81–84. Gomes, A., 2002. Aspectos da cadeia produtiva do couro bovino no Brasil e em Mato Grosso do Sul [WWW Document]. 2002. URL http://www.cnpgc.embrapa.br/publicacoes/doc/doc127/08aspectos.html Gomes, P.R., Koller, W.W., Gomes, A., Carvalho, C.J.B., Zorzatto, J.R., 2002. Dípteros fanídeos vetores de ovos de Dermatobia hominis em Campo Grande, Mato Grosso do Sul. Pesqui. Agropecu. Bras. 22, 114–118. Guimarães, J.H., Papavero, N., Prado, A.P., 1983. As miíases na região neotropical (identificação, biologia, bibliografia). Rev. Bras. Zool. 1, 239–416. Hill, S.K., Connelly, C.R., 2011. Human Bot Fly, torsalo (Central America), moyocuil (Mexico), berne (Brasil), mucha (Colombia), mirunta (Peru), and ura (Argentina, Paraguay, and Uruguay), Dermatobia hominis (Linnaeus , Jr.) (Insecta: Diptera: Oestridae) [WWW Document]. URL http://edis.ifas.ufl.edu/in775 (accessed 4.18.14).
Holdsworth, P.A., Vercruysse, J., Rehbein, S., Peter, R.J., De Bruin, C., Letonja, T., Green, P., 2006. World Association for the Advancement of Veterinary Parasitology (W.A.A.V.P.) guidelines for evaluating the efficacy of ectoparasiticides against myiasis causing parasites on ruminants. Vet. Parasitol. 136, 15–28. Hu, J.-M., Wang, C.-C., Chao, L.-L., Lee, C.-S., Shih, C.-M., 2013. First report of furuncular myiasis caused by the larva of botfly, Dermatobia hominis, in a Taiwanese traveler. Asian Pac. J. Trop. Biomed. 3, 229–31. Inforzato, G.R., Coelho Neto, E., Santos, W.R.M., Piccinin, A., Filadelpho, A. luis, 2008. Uso da ivermectina no combate do berne (Dermatobia hominis) em novilhas de cruzamento industrial. Rev. Científica Eletrônica Med. Veterinária 6, 1–5. Leite, R.C., Umehara, O., Caprioni Jr., L., Oliveira, P.R., Facury Filho, E.J., Okano, H., 1999. Efficacy and persistence of doramectin and ivermectin against natural infestations of Dermatobia hominis larvae in cattle. Rev. Bras. Parasitol. Veterinária 8, 161–166. Lopes, W.D.Z., Teixeira, W.F.P., Felippelli, G., Cruz, B.C., Maciel, W.G., Matos, L.V.S., Pereira, J.C.M., Buzzulini, C., Soares, V.E., Santos, T.R., Oliveira, G.P., Costa, A.J., 2013. Ivermectina e abamectina em diferentes doses e vias de aplicação contra larvas de Cochliomyia hominivorax em bolsas escrotais de bovinos recém-castrados, provenientes da região sudeste do Brasil. Ciência Rural 43, 2195–2201. Madeira, N., Amarante, A.F.T., Padovani, C., 2000. Diversity of ectoparasites in sheep flocks in São Paulo, Brazil. Trop. Anim. Health Prod. 32, 225–232. Maia, A.A.M., Gomes, A.G., 1988. Vetores de Dermatobia hominis LINNAEUS JR., 1781 (DIPTERA: CUTEREBRIDAE) na região de Uberaba, Minas Gerais. Rev. da Fac. Med. Veterinária e Zootec. da Univ. São Paulo 25, 47–51. Marques, F.A.C., Yamamura, M.Y., Vidotto, O., 2000. Lesões no couro bovino causadas pelos principais ectoparasitas nas regiões noroeste do estado do Paraná e sudoeste do estado do Mato Grosso. Semin. Ciências Agrárias 21, 33–39. Monteiro, M.R., Prado, A.P., 2000. Ocorrência de Trichopria sp. (Hymenoptera: Diapriidae) Atacando Pupas de Chrysomya putoria (Wiedemann) (Diptera: Calliphoridae) na Granja. An. Soc. Entomológica Bras. 29, 159–167. Netto, F.S.G., Gomes, A., Magalhães, J.A., Tavares, A.C., Domingues, C.A., 2006. Efeito da avermectina no controle da Dermatobia hominis em Presidente Médici, Rondônia, Brasil. Rev. Electrónica Vet. 7, 1–5. Neves, J.H., Carvalho, N., Rinaldi, L., Cringoli, G., Amarante, A.F.T., 2014. Diagnosis of anthelmintic resistance in cattle in Brazil: A comparison of different methodologies. Vet. Parasitol. 206, 216–226.
Oliveira-Sequeira, T.C.G., Amorim, R.M., Borges, A.S., Brant, M.P.R., 2014. Eficácia Terapêutica e Residual de Seis Formulações Inseticidas sobre o Parasitismo por Larvas de Dermatobia hominis em Bovinos. Veterinária e Zootec. 21, 177–186. Reck, J., Klafke, G.M., Webster, A., Dall’Agnol, B., Scheffer, R., Souza, U.A., Corassini, V.B., Vargas, R., dos Santos, J.S., Martins, J.R.D.S., 2014. First report of fluazuron resistance in Rhipicephalus microplus: a field tick population resistant to six classes of acaricides. Vet. Parasitol. 201, 128–136. Rodríguez, B.Z., Leite, R.C., 1997. Ocorrência de Vetores Biológicos da Dermatobia hominis (L.Jr., 1781) (Díptera: Cuterebridae), Capturados com Armadilha Magoom na Região Metalúrgica do Estado de Minas Gerais, Brasil. Ciência Rural 27, 645–649. Silva, A.M., Alencar, M.M., Regitano, L.C.A., Oliveira, M.C.S., 2010. Infestação natural de fêmeas bovinas de corte por ectoparasitas na região sudeste do Brasil. Rev. Bras. Zootec. 39, 1477–1482. Silva-Junior, V.P., Leandro, A.S., Borja, G.E.M., 1998. Ocorrência do Berne, Dermatobia hominis (DIPTERA: CUTEREBRIDAE) em Vários Hospedeiros, no Rio de Janeiro, Brasil. Parasitol. al Dia 22. Verocai, G.G., Fernandes, J.I., Ribeiro, F.A., Melo, R.M.P.S., Correia, T.R., Scott, F.B., 2009. Furuncular myiasis caused by the human botfly, Dermatobia hominis, in the domestic rabbit: case report. J. Exot. Pet Med. 18, 153–155.
Table 1 – Number of Dermatobia hominis larvae in cattle 10 days after treatment with ivermectin or moxidectin. Number of larvae 0 1 2 3 4 7 Mean Standard Deviation Standard Error Efficacy (%)
No. of cattle – Pardinho Farm Ivermectin Moxidectin Control 4 2 6 3 5 2 1 2 1 1 0 1 1 0 0 0 1 0 1.20 1.60 0.70 1.40 2.01 1.06 0.44 0.64 0.33 NS NS -
No. of cattle – Anhembi Farm Ivermectin Moxidectin Control 8 9 8 2 0 0 0 0 1 0 0 0 0 1 1 0 0 0 0.20 0.40 0.60 0.42 1.26 1.35 0.13 0.40 0.43 67 33 -
NS = larvae count of the treated group was higher than that of the control group (i.e., zero efficacy). Ten animals per group were examined on each farm. Figure 1 – Musca domestica carrying Dermatobia hominis eggs in the lateral-ventral region of the abdomen. Red circles indicate characteristic structures of Musca domestica.
Figure 2 – Extraction of live Dermatobia hominis larvae from the subcutaneous tissue of cattle 10 days after the administration of injectable ivermectin.
Figure 1 .
Figure 2 .
S0304-4017(15)00324-6 http://dx.doi.org/doi:10.1016/j.vetpar.2015.06.029 VETPAR 7682
To appear in:
Veterinary Parasitology
Received date: Revised date: Accepted date:
27-4-2015 22-6-2015 23-6-2015
Please cite this article as: Neves, Jos´e Henrique das, Carvalho, Nadino, Amarante, Alessandro F.T., Dermatobia hominis: potencial risk of resistance to macrocyclic lactones.Veterinary Parasitology http://dx.doi.org/10.1016/j.vetpar.2015.06.029 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Dermatobia hominis: potencial risk of resistance to macrocyclic lactones
José Henrique das Nevesa*, Nadino Carvalhoa, Alessandro F. T. Amarantea
a
UNESP – Universidade Estadual Paulista Júlio de Mesquita Filho, Departamento de
Parasitologia, IBB, CEP 18618-970, Botucatu, SP, Brazil. *Corresponding author: [email protected]
Highlights
•
First report of Dermatobia hominis resistance to ivermectin.
•
Several larvae found to have survived the ivermectin treatment.
•
The effectiveness of moxidectin to control D. hominis was low.
Abstract
Dermatobia hominis is an ectoparasite that infests various species of mammals, including cattle, impairing the quality of cowhides and leather. After observing natural infestation with D. hominis larvae in cattle on two farms in the state of São Paulo, Brazil, we evaluated the efficacy of two macrocyclic lactones, ivermectin and moxidectin, against this parasite. The drugs were administered to 10 animals in each group, following the manufacturer’s instructions. The groups were: Group 1 – treated with ivermectin (0.2 mg/kg of body weight (BW)); Group 2 – treated with moxidectin (0.2 mg/kg BW); and Group 3 – control (untreated). On the farm in Pardinho, a total of 12 and 16 live larvae were found in 6 and in 8 animals 10 days after the treatment with ivermectin and moxidectin, respectively, while in the control group 4 bovines had a total of 7 live larvae. On the farm in Anhembi, 2, 4 and 6 live larvae were extracted from ivermectin, moxidectin and control groups, respectively, after the treatment. This
is the first report of the presence of live D. hominis larvae after the treatment of cattle with ivermectin and moxidectin in Brazil. Keywords: Drug resistance, ivermectin, macrocyclic lactones
1. Introduction Dermatobia hominis is an ectoparasite that affects several mammal species, such as pigs, goats, dogs, cats, humans (Silva-Junior et al., 1998), sheep (Madeira et al., 2000), rabbits (Verocai et al., 2009), cattle (Silva et al., 2010), and sometimes horses (Daemon and Prata, 1997). This ectoparasite, which also parasitizes humans (Hu et al., 2013), occurs from the southern North America to southern Argentina (Hill and Connelly, 2011). During flight, D. hominis adult flies capture phoretic flies, upon whose ventral abdominal region they deposit 4 to 52 eggs (Gomes et al., 2002; Maia and Gomes, 1988). There are reports of 46 different vectors, including mosquitoes, flies and ticks (Guimarães et al., 1983). After 4 to 6 days of incubation, a first instar larvae is formed inside the egg (Barbosa et al., 2002). The larvae actively penetrate the host’s skin and begin their development in the subcutaneous tissue (Leite et al., 1999). In experimental infestations of D. hominis in cattle, it was observed that, in the initial days after infestation, the holes in the skin that enable the larvae to breath are practically imperceptible, but 7 days after infestation, nodules become visible in the subcutaneous tissue. At around day 14 post-infestation, the larvae molt into the second larval stage (L2) and nodules are approximately 1 cm in size. The nodules progressively increase in size, reaching 2 to 3 cm in diameter by 30 days post-infestation. At this point, exudate is eliminated through the hole through which the L3 larvae breathe.
Between day 33 and 42 post-infestation, L3 larvae complete their development and begin to leave the host (Barbosa et al., 2002). Although some studies report losses resulting from combined (simultaneous) parasitism by the gastrointestinal nematodes, Haematobia irritans, Rhipicephalus microplus and D. hominis (Bianchin et al., 2007; Netto et al., 2006), the major financial loss caused by D. hominis infestations is the decrease in leather quality due to perforations (Gomes, 2002; Marques et al., 2000). Today, leather is the second largest export product of the Brazilian cattle industry, generating annual revenues of over 2.48 billion dollars (ANUALPEC, 2014). However, about 40% of the leather produced in Brazil has low commercial value due to injuries caused by ectoparasite infestations, and 89% of the leather produced in the states of Mato Grosso, Minas Gerais, Rio de Janeiro and Sao Paulo States has perforations caused by D. hominis (Gomes, 2002). In Brazil, endoparasite and ectoparasite infestations in cattle are controlled mainly with drugs, especially with the macrocyclic lactone class of endectocides. However, there have been frequent reports of anthelmintic resistance (Neves et al., 2014), and also reports of R. microplus (Reck et al., 2014) and Cochliomyia hominivorax (Lopes et al., 2013) resistance to treatment with macrocyclic lactones. This paper describes, for the first time, the presence of live D. hominis larvae after the treatment of cattle with ivermectin and moxidectin in Brazil.
2. Materials and methods This work was developed in accordance with the ethical principles of animal experimentation and was approved by the Ethics Committee on Animal Use of the School of Veterinary Medicine and Animal Science of UNESP at Botucatu (Protocol 44/2012/CEUA-FMVZ).
2.1. Cattle Herds and Management In February and March 2013, during an experiment conducted to evaluate anthelmintic resistance in cattle (Neves et al., 2014), we observed, by visual inspection, that most of the animals of two farms were presenting the typical lesions on skin caused by D. hominis infestation. For this reason, we decided to also evaluate the efficacy of macrocyclic lactones against D. hominis larvae in the cattle on those farms; one located in the municipality of Pardinho and one in the municipality Anhembi, both in the state of São Paulo, Brazil. The animals of the farm in Pardinho were Holstein heifers, with a mean age (± standard deviation) of 12.5±3.92 months. The animals of the farm in Anhembi were Nellore male calves, with a mean age of 8.26±0.66 months.
2.2. Experimental procedure The animals were weighed and separated into groups in order to evaluate the efficacy of anthelmintics (Neves et al., 2014). The drugs were administered to 10 animals in each group, given by subcutaneous injection following the manufacturer’s instructions. The groups were: Group 1 – treated with ivermectin (0.2 mg/kg of body weight (BW), Ivomec®, Merial, Brazil); Group 2 – treated with moxidectin (0.2 mg/kg BW, Cydectin®, Fort Dodge, Brazil); Group 3 – control (untreated).
2.3. Physical examination of the animals Ten days after the drugs were administered, the animals were immobilized for a visual inspection and palpation of all the nodules over their entire bodies. The larvae were extracted by manual compression of the nodules, and checked to determine if they were moving, i.e., live, as recommended by the World Association for the Advancement
of Veterinary Parasitology (WAAVP) (Holdsworth et al., 2006). These procedures were photographed and filmed. The manual compression of the nodules was not performed on the day of the treatment to avoid damaging or killing the larvae, which could cause confusion with the efficacy of the drug treatment.
2.4. Statistical analysis The efficacy was calculated according to the following formula: 100 x [(CT)/C], to determine the geometric mean parasite density of the treated (T) and control (C) animals. For a therapeutic claim a reduction of larvae counts should be at least 90%, calculated by Abbott’s formula (Abbott, 1925) to compare treated and control animal groups (Holdsworth et al., 2006).
3. Results 3.1. History of use and criteria for the choice of anthelmintics The manager of the farm in Pardinho reported that the body weight of the animals was recorded prior to the administration of endectocides in order to determine the correct dosage of the drugs, according to manufacturer’s recommendation. Ivermectin, abamectin or doramectin were administered six times per year. The manager also reported that he had administered a combination of pour-on abamectin + fluazuron (3.0 mg/kg fluazuron + 0.5 mg/kg Abamectin, Fluatac Duo®, Ouro Fino, Brazil) about 60 days prior to the experiment. The owner of the farm in Anhembi reported that the calculation of the dosage of endectocides was based on a visual estimate of the animals’ body weight. Ivermectin or moxidectin were administered 3 times a year. In addition, Fentione (15 mg/kg;
Tiguvon® 15 Spot-On®, Bayer Animal Health, Brazil) was applied to control D. hominis whenever the parasite was detected visually.
3.2 Capture of a Dipterous Vector During the collection of data in the farm in Pardinho, large numbers of flies were observed in the environment and on the animals. In addition a fly bearing eggs of D. hominis was captured and placed in a jar with a lid.
The specimen, which was
identified as Musca domestica based on its morphological wing and antenna structures, was carrying 24 eggs of D. hominis attached to the lateral-ventral region of its abdomen (Figure 1).
3.3 Efficacy against Dermatobia hominis The efficacy of moxidectin and ivermectin on the farm in Pardinho was zero (Table 1), i.e., the count of viable larvae (Figure 2) recovered from the groups treated with moxidectin or ivermectin was higher than in the control group. At this farm, 3 bovines showed one live larvae, while other 3 animals presented two, three or four live larvae, after treatment with ivermectin (Table 1). Of the 10 animals treated with moxidectin, eight had live larvae: five animals with one larvae; two animals with two larvae; and one bovine with seven larvae. In the control group 4 bovines had a total of 7 live larvae (Table 1). On the farm in Anhembi, the degree of infestation was lower: 2, 4 and 6 live larvae were extracted from cattle of the ivermectin, moxidectin and control groups, respectively, corresponding to an efficacy of 67% and 33% (Table 1).
4. Discussion
Despite the relatively small number of larvae parasitizing the animals, several larvae were found to have survived the treatment. The efficacy of ivermectin in cattle infested with D. hominis is considered very high, with reports of 100% efficacy 7 to 10 days post treatment (Inforzato et al., 2008; Oliveira-Sequeira et al., 2014). Therefore, this is the first study in which live D. hominis larvae was found after treatment of cattle with ivermectin. The effectiveness of moxidectin to control D. hominis was also low. However, this anthelmintic does not have a registered statement of effectiveness against D. hominis, although it does against Hypoderma sp., which is a parasite not found in Brazil. The resistance of D. hominis to ivermectin is a very important issue because this drug is used extensively on Brazilian cattle (Neves et al., 2014). It is assumed that, with the continued use of the product, resistance to it will spread across the country, with negative consequences to the leather industry. The diptera (M. domestica) that was carrying the eggs of D. hominis has already been described as a vector in the state of Minas Gerais (Rodríguez and Leite, 1997). However, this is the first time M. domestica was found bearing eggs of D. hominis in the state of São Paulo. It should be noted that M. domestica is a synanthropic fly that occurs abundantly throughout the state of São Paulo and, according to Monteiro and Prado (2000), it can be found all year round.
Conclusion This is the first report of live D. hominis larvae found after the treatment of cattle with macrocyclic lactone (ivermectin), indicating development of resistance in such species.
Acknowledgements José Henrique das Neves and Nadino Carvalho gratefully acknowledge the financial support of CAPES (Brazil’s Federal Agency for the Support and Improvement of Higher Education) and Alessandro F. T. Amarante acknowledges the support of CNPq (Brazil’s National Council for Scientific and Technological Development).
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Table 1 – Number of Dermatobia hominis larvae in cattle 10 days after treatment with ivermectin or moxidectin. Number of larvae 0 1 2 3 4 7 Mean Standard Deviation Standard Error Efficacy (%)
No. of cattle – Pardinho Farm Ivermectin Moxidectin Control 4 2 6 3 5 2 1 2 1 1 0 1 1 0 0 0 1 0 1.20 1.60 0.70 1.40 2.01 1.06 0.44 0.64 0.33 NS NS -
No. of cattle – Anhembi Farm Ivermectin Moxidectin Control 8 9 8 2 0 0 0 0 1 0 0 0 0 1 1 0 0 0 0.20 0.40 0.60 0.42 1.26 1.35 0.13 0.40 0.43 67 33 -
NS = larvae count of the treated group was higher than that of the control group (i.e., zero efficacy). Ten animals per group were examined on each farm. Figure 1 – Musca domestica carrying Dermatobia hominis eggs in the lateral-ventral region of the abdomen. Red circles indicate characteristic structures of Musca domestica.
Figure 2 – Extraction of live Dermatobia hominis larvae from the subcutaneous tissue of cattle 10 days after the administration of injectable ivermectin.
Figure 1 .
Figure 2 .