USO DE COMPOSTOS REPELENTES PRODUZIDOS POR CÃES RESISTENTES AO Rhipicephalus Sanguineus Sensu Lato PARA O SEU CONTROLE EM CÃES SUSCEPTIVEIS

UNIVERSIDADE FEDERAL DE GOIÁS ESCOLA DE VETERINÁRIA E ZOOTECNIA PROGRAMA DE PÓS-GRADUAÇÃO EM CIÊNCIA ANIMAL

USO DE COMPOSTOS REPELENTES PRODUZIDOS POR CÃES RESISTENTES AO Rhipicephalus sanguineus sensu lato PARA O SEU CONTROLE EM CÃES SUSCEPTIVEIS

Jaires Gomes de Oliveira Filho Orientadora: Drª. Lígia Miranda Ferreira Borges

GOIÂNIA 2018 ii

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JAIRES GOMES DE OLIVEIRA FILHO

USO DE COMPOSTOS REPELENTES PRODUZIDOS POR CÃES RESISTENTES AO Rhipicephalus sanguineus sensu lato PARA O SEU CONTROLE EM CÃES SUSCEPTIVEIS

Tese apresentada para a obtenção do grau de Doutor em Ciência Animal junto à Escola de Veterinária e Zootecnia da Universidade Federal de Goiás

Área de Concentração: Saúde Animal, Tecnologia e Segurança de alimentos

Linha de Pesquisa: Parasitologia e doenças parasitárias dos animais de companhia, produção e selvagens

Orientadora: Profª Drª Lígia Miranda Ferreira Borges – IPTSP/UFG Comitê de Orientação: Profª Dr Éverton Kort Kamp Fernandes – IPTSP/UFG Profª Drª Mara Cristina Pinto – Unesp/Araraquara

GOIÂNIA 2018 iv

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Dedico este trabalho ao meu pai Jaires Gomes de Oliveira Que sempre acreditou no meu potencial e sempre me incentivou dar meu melhor (In memoriam)

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AGRADECIMENTOS

Em primeiro lugar à Profª Drª. Lígia Miranda Ferreira Borges, a quem serei eternamente grato por abrir as portas de seu laboratório para mim, me acolher e me orientar no início de minha jornada acadêmica. Acima de tudo pela paciência, presteza e compreensão que sempre guiou suas orientações e correções, e pelo exemplo de integridade e ética na pesquisa. Aos pesquisadores Dr. Michael A. Birkett e Dr. Jonh A. Pickett pela receptividade e acolhida no Rhothamsted research lugar no qual tive a grande oportunidade de aprender e desenvolver novas técnicas. Também ao grande colega Dr. André Lucio Sarria Franceschini que sempre esteve ao meu lado me orientando e guiando durante meu período no instituto, e a quem tenho a honra de chamar de meu grande amigo. A todos os colegas que passaram, estão e também aos que chegaram ao Laboratório de Parasitologia Veterinária da UFG e ao setor de Medicina Veterinária Preventiva, pela ajuda, conselhos, coleguismo e prestatividade. Principalmente as minhas amigas de mestrado e doutorado Lorena Lopes Ferreira, Adriana Marques Faria, Luiza Gabriella Ferreira de Paula e Fernanda de Oliveira Silva que sempre me ajudaram durante toda a condução dos experimentos e em toda a caminhada até aqui. Ao Dr. Gabriel Moura Mascarin quem tive a grande honra de conhecer durante meu período na pós graduação, que com certeza nos ajudou e nos conduziu em muitas dúvidas. À Escola de Veterinária e Zootecnia da UFG, por disponibilizar a estrutura para a execução dos experimentos, assim como pela oportunidade ímpar de participar de seu programa de pós-graduação. Também aos seus funcionários e prestadores de serviços pela colaboração e assistência. A Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES), pela concessão da bolsa de estudos, assim como ao Conselho Nacional de Desenvolvimento Tecnológico e Cientifico (CNPq) pelo financiamento do projeto sem o qual seria impossível a execução destes trabalhos. Principalmente a minha família, meus pais Jaires Gomes de Oliveira (In memoriam) e Iria Inês Gomes Vilela e minha irmã Naiara Cristina Gomes Vilela, que sempre estiveram ao meu lado tanto nos momentos de alegria quanto nos momentos de tristeza e que foram meu esteio durante toda minha vida inclusive nesta jornada acadêmica.

“Todas as vitórias ocultam uma abdicação”. (Simone de Beauvoir) 1908-1986

SUMÁRIO

CAPÍTULO 1: Considerações Gerais...... 1 1 INTRODUÇÃO ...... 1 2 REVISÃO DE LITERATURA...... 2 2.1. Rhipicephalus sanguineus sensu lato o carrapato marrom do cão...... 2 2.2. Busca por hospedeiros ……………………………………………………………….…5 2.3. Controle de carrapatos e o problema da resistência...... 6 2.4. Repelentes...... 7 2.5. Aposematismo olfativo: A rejeição de hospedeiros resistentes por carrapatos mediante a percepção de compostos voláteis presentes neste...... 9 2.6. Uso de compostos voláteis de não-hospedeiros (non-host) para o controle de artrópodes...... 13 3 OBJETIVOS...... 16 3.1. Objetivos Gerai ...... 16 3.2. Objetivos Especificos ...... 16 REFERÊNCIAS...... 17 CAPÍTULO 2: Quantification of brown dog tick repellents, 2-hexanone and benzaldehyde, and release from tick-resistant beagles, Canis lupus familiaris...... 26 Resumo...... 26 Abstract...... 26 Introduction...... 27 Material and Methods...... 28 Odor collection…………………………………………………………………………….28 Gas chromatography (GC) and coupled GC–mass spectrometry (GC–MS) analysis………29 2-hexanone and benzaldehyde quantification...... 29 Results ...... 31 Quantitative analysis of tick repellent compounds Estimation of the quantity of 2-hexanone and benzaldehyde produced by the beagles ...... 34 Discussion ...... 35 Acknowledgments ...... 36 References...... 37 xi

CAPÍTULO 3: Brown dog tick, Rhipicephalus sanguineus sensu lato, infestation of susceptible dog hosts is reduced by slow release of semiochemicals from a less susceptible host...... 41 Resumo...... 41 Abstract...... 42 Introduction...... 42 Material and methods...... 44 Animals…………...... 44 Ticks...... 44 Preparation and use of prototype non-host semiochemical delivery system………………..45 Tick infestation, evaluation, and sampling…………………………………………………45 Statistical analysis………………………………………………………………………….46 Results...... 47 Discussion...... 51 Acknowledgments………………………………………………………………………...54 References...... 54 CAPÍTULO 4: Persistence and efficacy of a new formulation based on dog allomonal repellents against Rhipicephalus sanguineus sensu lato tick ...... 61 Resumo………………………………………………………………………………...….61 Abstract…………………………………………………………………………………...62 Introduction……………………………………………………………………………....62 Material & Methods……………………………………………………………………...64 Results………………………………………………………………………..……………66 Discussion………………………………………………………………………..………..70 Conclusion……………………………………………………………………….………..71 Acknowledgments…………...... ………………….72 References……………………………………...... …72 CAPÍTULO 5: Considerações Finais …………………………………………………...75 xii

LISTA DE FIGURAS

Figure 1 (cap1) Co-injection of Beagle odour extracts with authentic standards 2- hexanone(0.033 ng mL−1) and benzaldehyde (0.016 ng mL−1); (A): synthetic compounds; (B):Dichloromethane extract from beagle 1 day 0; (C): Co-injection of beagle samplewith synthetic 2-hexanone and benzaldehyde. The peak for 2-hexanone is labelledas 1; the peak for benzaldehyde is labelled as 2……...... 32 Figure 2 (cap 1)The GC and GC/MS of DCM extracts of the odours of the three beagles, in all four days evaluated. The 12 spectra were highly similar, and 2-hexanone and benzaldehyde were present in all extracts. A1: Beagle 1 day 0; A2: Beagle 1 day 1; A3: Beagle day 4; A4: Beagle day 7; B1: Beagle 2 day 0; B2: Beagle 2 day 1; B3: Beagle 2 day 4; B4: Beagle2 day 7; C1: Beagle 3 day 0; C2: Beagle 3 day 1; C3: Beagle 3 day 4; C4: Beagle 3 day 7; the peak for 2-hexanone is indicated labelled as 1; and the peak for Benzaldehyde is labelled as 2………...... 32 Figure 1 Mean (cap2) (±SE, standard error) number of ticks (larvae, nymphs, and adults) retrieved from untreated (control) and treated (repellent collar) dogs across infestation periods. Vertical arrows indicate when dogs received the repellent collar. At4th infestation interval, the repellent collar was replaced by a new one and in the 5ththe repellent collar was removed. The counts during a week, except the first count during 10 days. Significant difference between control and treated dogs is indicated by P < 0.05 (*) or P < 0.01 (**)...... 49 Figure 2 (cap 2) Overall mean (±SE, standard error) number of ticks (larvae, nymphs, and adults) retrieved from untreated (control) and treated (repellent collar) dogs. Significant difference between control and treated dogs is indicated by P < 0.05 (*) or P < 0.01 (**)...... 50 Figure. 1. (cap 3) Mean (±SE, standard error) number of ticks (larvae, nymphs, and adults) retrieved from untreated (control) and treated (repellent collar) dogs across infestation periods. The repellent collars were attached on the 2nd week and dogs used them for 4 weeks (indicated by vertical arrows). Individual data points were shown per week. Significant difference between control and treated dogs is indicated by P < 0.05 (*) or P < 0.01 (**)…67 Figure (cap3). 2. Overall mean (±SE, standard error) number of ticks (larvae, nymphs, and adults) retrieved from untreated (control) and treated (repellent collar) dogs. Individual data xiii

points are shown. Significant difference between control and treated dogs is indicated by P < 0.05 (*) or P < 0.01 (**)…………………………………………………………………69

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LISTA DE TABELAS

Table 1 (cap1)Concentration of 2-hexanone and benzaldehyde in odour samples of three beagles across four days during one week, having analysed samples by GC and conversion of peak areas from the spectra into concentrations (ng mL−1) via linear regression models (see Tables 3 and 4). Beagles 1 and 3 are female, beagle 2 is male. There were no significant (p < 0.05, F-tests) effects of time or between beagles………...... 33 Table 2 (cap1) Ratio of benzaldehyde to 2-hexanone in odour samples of three beagles across four days during one week. Beagles 1 and 3 are female, beagle 2 is male. There was some evidence of real difference between beagles (p = 0.057, F-test), the standard error of the difference between the means for the beagles was 0.506 on 6◦ of freedom 2 ……...... 33 Table 3 (cap1)Summary of the output of the ANOVA for the linear regression analysis of 2- hexanone. The degrees of freedom (df), sums of squares (SS), mean squares (MS), variance ratio (VR) and p-value for the F-test of the variance ratio (F.pr.) are given along with the estimated coefficients in the linear regression model, their standard errors (SE), partial t- statistics on 13 df and p-values for the t-tests of these t-statistics...... 33 Table 4 (cap1) Summary of the output of the ANOVA for the linear regression analysis of benzaldehyde. The degrees of freedom (df), sums of squares (SS), mean squares (MS), variance ratio (VR) and p-value for the F-test of the variance ratio (F.pr.) are given along with the estimated coefficients in the linear regression model, their standard errors (SE), partial t-statistics on 13 df and p-values for the t-tests of these t-statistics...... 33 Table 1 Mean (cap2) (±SD) of biological parameters of Rhipicephalus sanguineus sensu lato ticks obtained along successive infestations on dogs treated or non- treated with 2- hexanone and benzaldehyde...... 50

ANEXOS

Anexo I – Quantification of brown dog tick repellents, 2-hexanone andbenzaldehyde, and release from tick-resistant beagles, Canis lupus familiaris. Journal of Chromatography B, 1022 (2016) 76. Anexo II – Brown dog tick, Rhipicephalus sanguineus sensu lato, infestation of susceptible dog hosts is reduced by slow release of semiochemicals from a less susceptible host. Ticks and Tick-borne Diseases 8 (2017) 77. Anexo III - Parecer de Aprovação no Comitê de Ética 78-79.

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RESUMO

Rhipicephalus sanguineus sensu lato, também conhecido como “carrapato marrom do cão”, parasita preferencialmente o cão doméstico, no entanto pode parasitar inúmeras outras espécies mamíferas inclusive seres humanos. Este carrapato demonstra um comportamento de busca ativa por seu hospedeiro, sendo amplamente atraído pelo latido e pelo CO2 e outros sinais relacionados a presença de seus hospedeiros. Apesar de parasitar preferencialmente cães sabe-se que algumas raças como o beagle são menos parasitadas que outras raças como Cocker spaniel inglês que podem apresentar uma carga parasitaria cerca de até 11,5 vezes maior que cães mestiços. É notável a capacidade de carrapatos e demais artrópodes tem em selecionar seus hospedeiros em relação a características desejáveis ou indesejáveis que conseguem perceber destes, por meio da quimiorrecepção de compostos voláteis emanados pelos hospedeiros. A esta ferramenta adaptativa damos o nome aposematismo olfativo que é capacidade de um de parasito em evitar seus hospedeiros mediante a percepção de um químico emanado por este que sinalize uma resposta adaptava negativa ao artrópode. Geralmente esta resposta pode estar ligada a fatores imunes, excesso de grooming ou inacessibilidade ou demais fatores. Um dos grandes problemas na atualidade sem dúvida e o controle de carrapatos e doenças que são transmitem a seus hospedeiros durante o repasto sanguíneo. Uma das alternativas para o controle de parasitismo afim de dissuadir os carrapatos da busca por seus hospedeiros é uma utilização de compostos encontrados em hospedeiros resistentes que são interpretados por estes artrópodes como não hospedeiros. Estes compostos demonstram atividade mais especifica entre artrópode parasitos e seus hospedeiros, pois foram selecionados durante uma adaptação entre espécies envolvidas nesta relação parasito-hospedeiro forjando bases da seleção de parasitos por hospedeiros suscetíveis e resistentes.

PALAVRAS-CHAVES: Carrapato marrom do cão; repelentes; controle de carrapatos; aposematismo

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ABSTRACT

Rhipicephalus sanguineus sensu lato, the "brown dog tick", preferentially parasite the domestic dog, in the latter can parasite others mammals species including humans. This tick demonstrates a hunting behavior by its host, being widely attracted by the bark and the CO2 among other signs related to the presence of its hosts. Despite being commonly found parasite on dogs, it is known that some breeds such as beagles are less parasitic than other breeds like English Cocker Spaniel which can present a parasitic load about up to 11.5 times higher than mongrel dogs. It is notable a capacity of ticks and other arthropods in their hosts in relation to desirable or undesirable characteristics that can perceive these by means of chemoreception of volatile compounds emanated by these hosts. This adaptive tool is called olfactory aposematism. Olfactory apostotism is capable of an ectoparasite in avoiding its hosts through a perception of a volatile chemical emanated by it that signals a negative adaptive response to the arthropod. Usually this response may be linked to immune factors, excessive grooming, inaccessibility or other factors. One of the big problems nowadays undoubtedly is the control of ticks and diseases that are transmitted to their hosts during the blood repast. One of the alternatives for control of parasitism in order to deter ticks from the search for their hosts is a use of compounds found in resistant hosts that are interpreted by these arthropods as non-hosts. These compounds demonstrate a more specific activity between arthropod parasites and their hosts, since they were selected during an adaptation between species involved in this parasite-host relationship forging bases of parasite selection by susceptible and resistant hosts.

KEYWORDS: Brown dog tick; Repellents; Ticks Control; Aposematism

CAPITULO 1: CONSIDERAÇÕES GERAIS

1. INTRODUÇÃO

Os artrópodes, parasitos de hábitos hematófagos, debilitam e emaciam seus hospedeiros devido a seus hábitos de alimentação. Além disso, estes ectoparasitos transmitem uma infinidade de patógenos como vírus, bactérias, protozoários, ricketsias entre outros. Estes patógenos veiculados são um dos maiores problemas na atualidade tanto para a saúde pública quanto para economia mundial, devido ao grande poder de dispersão e colonização de novos ambientes destes artrópodes vetores1,2. Os carrapatos são um dos grupos de artrópodes hematófagos de grande importância pois possuem uma ampla distribuição global e inúmeras espécies1. Uma das espécies de grande interesse médico e veterinário é o Rhipicephalus sanguineus sensu lato, sendo o carrapato com maior distribuição geográfica dentre as espécies conhecidas, estando presente entre as latitudes 35ºS a 50ºN3. Também conhecido como o “carrapato marrom do cão” ou ainda “carrapato do canil”, seu hospedeiro natural, mas não exclusivo é o cão doméstico. As populações caninas são as principais fontes de manutenção e responsáveis por explosões populacionais desses parasitos no ambiente4,5, podendo este carrapato ainda parasitar animais de fazenda, cães selvagens, aves rasteiras inclusive seres humanos6-11. R. sanguineus s. l. é responsável pela transmissão de patógenos que causam doenças graves tanto em humanos como em animais1. Tendo em vista a importância de R. sanguineus s. l. faz-se necessário seu controle. Destaca-se que a principal forma de controle deste parasito ainda é o uso de acaricidas com bases químicas neurotóxicas, que ocasionam o surgimento de cepas resistentes12-14. Supõe-se que a formulação de novas bases químicas acaricidas e a manutenção de uma vigilância governamental sobre a venda destes produtos poderiam resolver o problema da resistência. No entanto, pesquisas para a formulação e avaliação da eficácia de novos acaricidas são onerosas e demoradas15,16. Este carrapato demonstra um comportamento de busca ativa por seu hospedeiro, 1,4 sendo amplamente atraído pelo latido e pelo CO2 . Podendo ainda responder positivamente ou negativamente a demais estímulos sonoros, térmicos, olfativos e táteis durante a busca por seus hospedeiros17-19. Sem dúvida os artrópodes, principalmente carrapatos, são as 2

espécies animais que têm os seus comportamentos mais influenciados pela percepção de compostos químicos1,20. Sendo, portanto, o estudo de químicos voláteis presentes em hospedeiros susceptíveis e resistentes uma ferramenta de controle para estes parasitos20. Estes compostos demonstram atividade mais especifica entre artrópode parasitos e seus hospedeiros, pois foram selecionados durante a adaptação entre espécies envolvidas nesta relação parasito- hospedeiro forjando bases da seleção de parasitos por hospedeiros suscetíveis e resistentes20- 22. Sendo que o uso destes métodos alternativos como estes compostos químicos voláteis que fazem parte da ecologia destes artrópodes vem ganhando destaque19,23-25.

2. REVISÃO DE LITERATURA

2. 1. Rhipicephalus sanguineus sensu lato - o carrapato marrom do cão

Carrapatos são artrópodes da classe Arachnida e subordem Ixodida. Atualmente são conhecidas cerca de 900 espécies, que se encontram divididas em três famílias, Ixodidae, Argasidae e Nuttalliellidae26,27. Os membros da família Ixodidae são os mais estudados, devido a sua importância na saúde pública e veterinária, além de prejuízos associados a produção animal1,28. Dentre os membros da família Ixodidae se destaca o grupo de espécies conhecido como Rhipicephalus sanguineus, o carrapato marrom do cão, que possui ampla distribuição global29,30. Rhipicephalus sanguineus strictu sensu foi originalmente descrito como um carrapato presente em cães habitando a região da Galia, o que a grosso modo representa a França, Bélgica e Itália nos dias atuais3,30. Primeiramente classificado como Ixodes sanguineus em 1806 por Latreille, seu neótipo foi perdido o que dificulta a classificação de espécies deste grupo30, uma vez que estas espécies têm predileção por parasitarem cães e são morfologicamente semelhantes a olho nu31,32. A filogenia do grupo R. sanguineus vem sendo intensivamente investigada através de métodos de análise moleculares, atualmente sabe-se que apesar de várias espécies e subespécies terem sido classificadas originalmente como R. sanguineus s. s., existem ao menos duas linhagens bem definidas e distintas tanto geneticamente quanto morfologicamente, a linhagem temperada e a linhagem tropical31,33,34. Devido a correlação genética próxima entre espécimes encontrados em zonas temperadas sugere-se que estes deveriam ser denominados como R. sanguineus s. s., sendo o local do

neótipo a França local de clima tipicamente temperado33,35. No entanto, mesmo nesta região espécimes de R. sanguineus coletados de cães mostraram divergência genética32. Consequentemente, o termo "R. sanguineus sensu lato", até que a avaliação morfológica e genética de carrapatos Rhipicephalus sanguineus de diferentes regiões geográficas seja concluída deve ser utilizado36. R. sanguineus s. l. provavelmente evoluiu como um parasito de canídeos em climas quentes e com a domesticação do cão e por conseguinte proximidade com o ser humano, passou a colonizar com facilidade ambientes intra-domiciliares3. Seu hospedeiro preferencial são cães domésticos4,5, mas pode parasitar inúmeros outros hospedeiros como animais de produção, pombos, canídeos selvagens e inclusive o homem6-11. Atualmente encontra-se distribuído em todo o território nacional, onde credita-se a introdução da espécie às primeiras incursões europeias que traziam consigo animais de companhia, por volta do século XVI7. Portanto, a sua atual prevalência, principalmente em regiões tropicais e subtropicais é resultado da atividade antrópica. R. sanguineus s. l. possui grande significância para a saúde pública e animal, uma vez que atua como vetor de inúmeros patógenos como vírus, bactérias e protozoários tanto para cães quanto para seres humanos1. É o principal vetor de Babesia vogeli, Erlichia canis e Anaplasma platys, hemoparasitos com altas taxas de prevalência em cães infestados por estes carrapatos1,37,38, podendo ainda atuar na transmissão de Hepatozoon canis39. R. sanguineus s. l. vem sendo incriminado na transmissão de Rickettsia rickettsii agente causal da febre maculosa para seres humanos principalmente em estados do sul dos Estados Unidos da América e no México40-42, no Brasil seu envolvimento no ciclo epidemiológico da doença é sugerido, principalmente em regiões urbanas, contudo o principal vetor neste pais ainda são carrapatos do gênero Amblyomma43,44. R. sanguineus s. l. atua ainda como vetor de Rickettsia conorii e Rickettsia massiliae ambos agentes patogênicos que podem levar ao óbito45,46. Contudo, a transmissão destes agentes patogênicos está ligada a origem do espécime, sendo que dentro das linhagens, temperada e tropical, existem espécimes com capacidades vetoriais diferentes32,38,39. Este é um carrapato trioxeno, ou seja, necessita para seu completo desenvolvimento três hospedeiros e realiza a muda no ambiente, apresentando quatro estágios de desenvolvimento, sendo ovo embrionado, larva, ninfa e adultos. Há duas fases distintas, a de vida livre e a parasitária, sendo estas fases complementares e fundamentais

para o desenvolvimento do ciclo do parasito, o qual utiliza o ambiente não apenas para ovipor mas também para fazer as ecdises1. As fêmeas desta espécie se alimentam por aproximadamente 10 dias, após serem fertilizadas pelos machos sexualmente maduros e em seguida, deixam o hospedeiro para a oviposição. Durante o período de pré-oviposição, que pode variar entre três a 14 dias, a fêmea busca ninhos próximos a seus hospedeiros. A oviposição pode durar até 18 dias e uma fêmea pode ovipor de 3.000 até 7.000 ovos dependendo de seu peso corporal inicial. O período de incubação dos ovos pode chegar até a 25 dias, larvas e ninfas quando fixadas em seus hospedeiros alimentam-se por três a cinco dias e cinco a sete dias, respectivamente47-49. Os machos podem permanecer durante semanas fixados a seus hospedeiros sem se destacar50, e após o repasto sanguíneo e maturação sexual chegam a deixar hospedeiros em busca de fêmeas51,52. Este fato aumenta o potencial e capacidade vetorial deste estágio51. Após se alimentarem, fêmeas e ninfas apresentam um ritmo de queda do hospedeiro semelhante, durante a escotofase, enquanto as larvas apresentam uma queda mais acentuado na fotofase53. Isto pode gerar uma vantagem estratégica na colonização de ambientes por parte de R. sanguineus s. l. visto que fêmeas ingurgitadas são o estágio com maior capacidade de colonização3,53. Uma vez fora de seus hospedeiros, larvas e ninfas ingurgitadas fazem a muda em um período aproximado de oito a 13 dias e de 11 a 25 dias, respectivamente, sob condições ideais48-50. O ciclo de vida parasitário é diretamente influenciado pelo hospedeiro em que o carrapato se fixa, sendo que o status imune geral do hospedeiro pode favorecer ou desfavorecer a evolução deste parasito47,48. Enquanto o ciclo de vida livre é diretamente influenciado por características abióticas como temperatura, umidade entre outros, podendo causar a dessecação rápida de carrapatos ou um estado de dormência temporário conhecido como diapausa4,50,54. Pesquisadores questionavam até então se R. sanguineus s. l. passava por diapausa já que este carrapato apresentava dinâmicas sazonais diferentes. Por exemplo, em regiões de clima temperado este carrapato tende a diminuir suas atividades de parasitismo durante o inverno o que indica uma diminuição em sua dinâmica sazonal e uma provável diapausa55, enquanto que em regiões de clima tropical não se verifica essa diminuição de atividade, sendo observada uma dinâmica com até quatro gerações completas4. Hoje sabe- se que a linhagem temperada desenvolve diapausa em climas frios, provavelmente como uma estratégia para esperar por hospedeiros, enquanto que a linhagem topical em climas quentes como os observados em locais tropicais não passa por diapausa54.

2.2. Busca por hospedeiros

Artrópodes reconhecem e interagem com o meio e outros animais através de sinais que desse conseguem captar, a essa interação denomina-se ecologia56,57. A importância que um sinal, seja ele acústico, físico, químico ou visual tem para cada espécie de artrópode está ligada diretamente a sua biologia comportamental e fisiologia58,59. Diferentemente de outros artrópodes hematófagos, que possuem um aparato visual desenvolvido para detecção de sinais relacionados a fontes de alimentação e oviposição, os carrapatos possuem uma morfofisiologia ímpar1,59. A maioria das espécies de carrapatos não possuem olhos propriamente ditos, mesmo aquelas que possuem, como algumas espécies do gênero Amblyomma é duvidoso que os olhos os possibilitem enxergar formas definidas, discriminar objetos e tampouco interpretar estes como um sinal da presença de um hospedeiro1. Soma-se a isto o fato de que carrapatos em geral não são artrópodes de grande mobilidade e dispersão60. Portanto, os carrapatos têm sua biologia comportamental voltada principalmente para a percepção de compostos químicos voláteis presentes no hálito, pele, digestão e glândulas de seus hospedeiros preferenciais61-63. Durante milhares de anos os carrapatos adaptaram tanto sua alimentação aos hospedeiros disponíveis, quanto sua quimiorrecepção para detecção e seleção de hospedeiros e até sítios de predileção para o repasto sanguíneo58,59,63-65. O órgão de maior importância para a percepção sensorial dos carrapatos é o órgão de Haller, presente em todas as espécies e localizado no dorso do tarso no primeiro par de patas1,66. Este órgão consiste de uma cápsula posterior, com numerosas sensilas expostas por meio de uma pequena abertura. As sensilas presentes neste órgão são diferenciadas pelo tipo de estímulos que estão aptas a captarem. Por exemplo, movimentos são detectados por mecanosensilas, como a direção ou mudanças na corrente de vento e, o calor, que é comumente emanado pela maioria dos vertebrados terrestres, é detectado por meio das termohigrosensilas, assim como variação de umidade. Porém as quimiossensilas apresentam um papel crucial na identificação dos hospedeiros, pois estas detectam os compostos químicos emanados por esses1,59,67. As quimiosensilas são ainda divididas em olfativas e gustativas, as olfativas são especializadas na percepção de compostos químicos voláteis como dióxido de carbono (CO2) e amônia

(NH3), enquanto sensilas gustativas são especializadas na percepção de compostos químicos encontrados em soluções como cloreto de potássio (KCl) e cloreto de sódio (NaCl)17,68.

Carrapatos são conhecidos por serem atraídos por fatores físicos como o calor, 67 além de compostos voláteis como CO2, sulfeto de hidrogênio (H2S) , acetona, oxido nítrico (NO)69, ácidos graxos, fenóis entre outros62. R. sanguineus s. l. é um carrapato que preferencialmente adota a estratégia de busca ativa por seus hospedeiros, em laboratório demonstra um comportamento anemotáxico positivo, ou seja, sempre busca caminhar contra a fonte de vento70,71. Demonstra grande atração a sinais relacionados com a presença de seus 1,4 hospedeiros preferenciais como CO2 e latidos , além de ser ativado e atraído em testes de laboratório por octenol, acido hexanoico e salicilato de metila71, responde positivamente à 72 NH3 em testes de eletrofisiologia . Porém, podem utilizar a emboscada como tática para localizar hospedeiros, se agregando em locais propícios para a localização destes, através da utilização do feromônio de arrestamento encontrado nas excretas, secreções e ecdises destes ectoparasitos. Como geralmente este material é encontrado próximo aos hospedeiros a identificação destes pontos como um local de agregação para estágios não alimentados fazerem emboscada confere uma vantagem estratégica para estes carrapatos1,73. Além disso, esta espécie de carrapato demonstra um comportamento de arrestamento mais acentuado quando odores de cães susceptíveis lhe são apresentados, que indica que este carrapato pode adotar esta tática de emboscada na busca por um hospedeiro preferencial18. Destaca-se ainda que o comportamento de R. sanguineus s. l. assim como de outros artrópodes hematófagos em relação à busca por hospedeiros pode variar tanto em função de fatores abióticos como fisiológicos51,59,67. Fatores abióticos como temperatura, umidade, velocidade e direção da pluma do vento, além de pressão atmosférica, interferem não apenas com a percepção dos químicos pelos artrópodes59,67, como também com a sua resposta comportamental74. Além de fatores fisiológicos, bióticos, como a busca por hospedeiros em mosquitos que é diretamente ligada com o ciclo gonadotrófico de fêmeas75. A busca pelo hospedeiro em machos de R. sanguineus s. l. pode estar também associada a uma maior movimentação deste estágio entre hospedeiros diferentes, quando, após sua maturação gonadotrófica, buscam por fêmeas para a cópula51,52.

2.3. Controle de Carrapatos e o Problema da Resistência

Tendo em vista a importância de R. sanguineus s. l. na saúde pública e em medicina veterinária é necessária a implementação de métodos de controle eficientes76,77. Apesar de inúmeros métodos terem sido explorados, principalmente experimentalmente, a

principal forma de controle deste carrapato ainda se baseia na utilização de acaricidas sintéticos, contudo há inúmeros relatos de resistência a estes compostos, mesmo em associações12-14. Sem dúvida, o desenvolvimento rápido de resistência por parte dos carrapatos, se deve tanto ao fato deste ser um artrópode que apresenta várias gerações anuais, quanto à pressão seletiva devido à interferência antrópica com o uso excessivo de bases químicas para o seu controle16. Pode-se destacar ainda que a busca e desenvolvimento por novas bases químicas com propriedades acaricidas é um processo longo e oneroso1,15,16. Além disso, provavelmente o potencial para o desenvolvimento de resistência em uma população alvo já esteja presente antes mesmo desta ser desafiada com o acaricida15. Isso se deve ao fato de na população pré-estabelecida existirem alguns indivíduos naturalmente resistentes, provavelmente devido a mutações heterozigóticas, a medida em que o uso do acaricida se estabelece, a população sensível é eliminada e acontece o efeito denominado de estabelecimento do alelo resistente. Os mecanismos fisiológicos da resistência estão relacionados, principalmente, com a diminuição de penetração cuticular do acaricida, a resistência metabólica devido ao aumento da detoxificação, e a resistência por insensibilidade do sítio de ação do acaricida1. Em contrapartida ao uso de acaricidas, cada vez mais utilizados e recomendados estão os métodos de proteção individual, como os repelentes. O uso de repelentes é recomendado como um item vital em áreas de surtos de doenças transmitidas por artrópodes. Sendo que estes apresentam como vantagens poderem ser aplicados a qualquer hora e local78. A repelência per se tem como intuito dissuadir o parasito de sua fonte de alimento, o hospedeiro79. Sendo uma estratégia que acaba tanto por diminuir a transmissão de patógenos quanto a própria população original de artrópodes vetores79,80.

2. 4. Repelentes

Em uma definição clássica, repelentes são qualquer estímulo que atue sob o comportamento de artrópodes afastando-os da fonte de estímulo atrativa. Podem ser classificados em repelentes físicos ou químicos, sendo que os químicos podem ser percebidos à distância como os olfativos (voláteis) ou por contato, como os gustativos (deterrentes)85. Em um conceito mais amplo os repelentes são compostos que atuam inibindo a localização de hospedeiros por artrópodes parasitos, portanto impedem sua alimentação e

logo sua proliferação, atuando em geral na fase de vapor79,80. Sendo ainda, no caso de carrapatos que são artrópodes que se fixam em seus hospedeiros para realizar a alimentação, definidos dois tipos de repelência: - sensu stricto a qual é caracterizada por um efeito irritante observado entre seis a oito horas após aplicação do composto, quando os carrapatos não fixados tendem a se afastar dos animais tratados; - sensu lato ou expelência, a qual ocorre 24 horas após a aplicação do repelente e estimula os carrapatos fixados a cessarem a alimentação e se “destacarem”77,86. O primeiro efeito, a repelência sensu stricto, pode ser atribuído à fase de vapor de um composto ou efeito irritante através do contato direto, observado com compostos como DEET (N,N-dietil-3-metilbenzamida) um repelente padrão, enquanto a expelência é atribuída à inibição, ou interrupção, da fixação devido a efeitos tóxicos do composto para o artrópode, geralmente observado com compostos com propriedades acaricidas reconhecidas como permetrina, fipronil e amitraz86. No contexto da saúde pública os repelentes desempenham um importante papel na proteção pessoal contra doenças transmitidas por vetores, pois impedem que os artrópodes hematófagos se alimentem evitando assim a propagação destes patógenos79-81. Por outro lado, a possibilidade de uso de repelentes para o controle de carrapatos em animais ainda é pouca avaliada. Kumar et al.82 avaliaram o efeito do DEET na fixação de R. sanguineus s. l. em coelhos observaram taxas de repelência significativas (90%) contra este carrapato para larvas, ninfas e adultos na concentração de 25 % de DEET. Sendo observado um efeito platô na duração da repelência ao se aumentar a concentração para 35-40%. Enquanto, Endris et al.83; Dryden et al.84 observaram que alguns compostos acaricidas causam um tipo de repelência, expelencia, por causarem a interrupção da alimentação e prevenção da fixação de carrapatos. Formulações de imidacloprid (8,8%) e permetrina (44,0%) são eficazes em prevenir a fixação e repelirem carrapatos Ixodes scapularis e Amblyomma americanum em cães tratados com estas por até três semanas84. Em tratamentos contra Ixodes ricinus a permetrina (65%) também demonstrou grande eficácia na repelência destes parasito e diminuição de sua fixação sobre cães tratados durante testes de duas horas de intervalo83. O pontencial de manipular o comportamento de artrópodes vetores pode ser explorado pelo desenvolvimento de repelentes com base em hipóteses que relacionam-se com a evolução da repelência, ressaltando-se basicamente três classes: compostos botânicos, compostos de espécies não-hospedeiras, e compostos de espécies hospedeiros20. Compostos botânicos podem interferir com a localização de hospedeiros por artrópodes hematófagos,

devido principalmente a resquícios evolucionários21,87. Espécies não-hospedeiras que são capazes de emitir uma maior quantidade voláteis e alguns voláteis específicos, sendo a resposta de artrópodes a estes compostos de espécies não-hospedeiras considerada de valor adaptativo mais alto que a compostos botânicos20,21. E ainda repelentes derivados de espécies hospedeiras, sendo observado que indivíduos de certas populações e espécies animais são bem menos parasitados que demais indivíduos, devido a emissão de alguns compostos exclusivos destes19,20.

2.5. Aposematismo olfativo: A rejeição de hospedeiros resistentes por carrapatos mediante a percepção de compostos voláteis presentes nestes

A pele dos vertebrados é conhecida por ser uma fonte emissora de inúmeros compostos químicos com diversas classes e estruturas diferentes, com padrões incomuns de ramificação e sítios de insaturação, contrastando com compostos de tecidos internos87-89. Tais compostos químicos presentes na pele de vertebrados podem ser reconhecidos por ectoparasitos como sinais de um hospedeiro não “desejável”, ou não hospedeiro ‘non-host’, a essa característica adaptativa podemos chamar de aposematismo olfativo21,22,87,90. O aposematismo olfativo se dá quando compostos químicos voláteis emitidos por uma presa, anunciam uma característica indesejável ao predador90, podendo ser extrapolado para a relação parasito-hospedeiro21,22. Em tal contexto, um artrópode parasito ao perceber um composto químico volátil emitido por um determinado hospedeiro que venha a lhe acarretar algum prejuízo em seu desenvolvimento evita o hospedeiro21. Em geral esse prejuízo está ligado a efeitos deletérios causados no desenvolvimento biológico do parasito pela imunocopetência do hospedeiro18,21,22,48. Ainda podendo estar ligada a outros fatores como o “grooming” excessivo, inacessibilidade ou desbalanço semioquimico21. Esta característica adaptativa dos artrópodes de evitar hospedeiros imunocompetentes devido a certos compostos que conseguem perceber, faz parte da co- evolução adaptativa entre parasitos e hospedeiros. A co-evolução é uma construção ativa entre indivíduos, como uma corrida armamentista, logo hospedeiros e parasitos interagem e transformam-se mutualmente91. Neste processo, hospedeiros mais susceptíveis são extintos, deixando apenas resistentes na população de hospedeiros91,92. Enquanto que os parasitos adaptam sua percepção sensorial para seleção e rejeição de hospedeiros que possam acarretar benefícios ou prejuízos ao seu desenvolvimento biológico22,90. Este processo é construído

durante milhares de anos, no entanto, se um parasito é muito virulento, ou extremamente agressivo, ele mata seu hospedeiro antes de completar sua prole, podendo reduzir tanto o número de parasitos altamente agressivos como aumentar o número de hospedeiros resistentes91. Um exemplo deste modelo de interação co-evolutiva é a dos bovinos zebuínos (Bos indicus) que apresentam menor susceptibilidade às infestações por Rhipicephalus (Boophilus) microplus que bovinos europeus (Bos taurus). Esta capacidade está ligada ao fato de zebuínos terem histórico de maior convivência com o R. microplus, haja visto que os dois têm origem asiática, enquanto que bovinos taurinos tiveram contato com este carrapato apenas após sua introdução nos trópicos92. Em testes comportamentais observa-se claramente um aumento no “questing” de larvas de R. microplus quando são expostas a odor de bovinos holandeses e 2-nitrofenol, composto presente em bovinos e que provavelmente contribui para especificidade desta espécie93. Também tiras adesivas contendo esfregaços da pele de bovinos susceptíveis demonstram maior eficácia na captura de larvas de R. microplus94. Contudo destaca-se que a seleção de hospedeiros imunidebilitados através da percepção de compostos voláteis presentes nestes também ocorre21. Observa-se que Ixodes hexagonus parasita significativamente mais ouriços terrestres (Erinaceus europaeus) acometidos por alguma patologia do que animais saudáveis. Esta atração ocorre devido a um composto volátil, o indol (C8H7N), presente exclusivamente em animais imunidebilitados por alguma doença95. Sendo que, alguns autores acreditam que a resposta imunológica é menos acentuada em relações de parasitismo já estabelecidas há muito tempo, como o R. sanguineus s. l. e cães (Canis lupus familiaris), levando a se acreditar que cães não teriam a capacidade de desenvolver nenhum grau de reação imune contra infestações de R. sanguineus s. l.47,96. Szabó47 observou que durante três sucessivas infestações em cães mestiços, carrapatos R. sanguineus s. l. recuperados destes animais obtiveram um aumento em parâmetros como peso da fêmea ingurgitada e conversão do peso corporal em ovos, nas duas últimas infestações. Bechara et al.96 tentaram induzir resistência ao R. sanguineus s. l. em cães mestiços, inoculando-os com três doses de extrato de carrapatos adultos não alimentados, no intervalo de 15 dias, seguido de infestação desafio 15 dias após a última imunização, os cães não demonstraram resistência significativa. Ferreira & Bechara97 realizaram trabalho comparando duas espécies canídeas, cães domésticos mestiços e cães-

do-mato Cerdocyon thous (LINNAEUS), as duas espécies canídeas foram submetidas a infestações sucessivas com carrapatos adultos. Durante as infestações sucessivas não houve diferença significativa entre os parâmetros biológicos dos carrapatos que foram observados. O grupo de cães-do-mato não apresentou resistência significativa, assim como cães domésticos. Recentemente um estudo visou comparar a capacidade de um grupo de cães da raça Dachshund em adquirir resistência a infestações sucessivas de carrapatos de cepas temperadas e tropicais de R. sanguineus s. l., observou-se que em três infestações sucessivas não houve diferença de parâmetros biológicos entre as cepas deste carrapato. Também no ensaio de imunoabsorção enzimática (ELIZA) não foi capaz de se notar um aumento significativo na titularidade sorológica de nenhum dos grupos49. Ressalta-se que em sua maioria os estudos mencionados acima foram feitos em cães sem raça definida (mestiços), e que ainda é preciso lembrar que a seleção artificial de cães vem sendo feita desde sua domesticação, a cerca de 500 mil anos, e ao se selecionar características desejáveis há uma correlação negativa de inúmeros fatores indesejáveis, um destes fatores pode ser a maior susceptibilidade a ectoparasitos como os carrapatos92,98. Características que definem a imunidade variam entre espécies, raças e até indivíduos20,92,99, sendo ainda que pode-se afirmar que tais características de imunidade possam ter sido adaptadas durante o processo de seleção de raças91,92,98. Louly et al.4 em trabalho de dinâmica sazonal de R. sanguineus observaram que cães da raça Cocker spaniel inglês apresentavam uma carga parasitária que chegava a ser 11,5 vezes maior que a de outros cães que coabitavam com estes. Em levantamento recente de carrapatos em cães de diversas raças constatou-se diferença no grau de parasitismo entre as raças avaliadas, sendo o Cocker spaniel Inglês a raça com maior grau de infestação por R. sanguineus s. l.100 Inokuma et al.101 observaram que cães da raça beagle eram capazes de adquirir resistência a R. sanguineus s. l. após sucessivas infestações, com diminuição efetiva do número de fêmeas ingurgitadas sobre esses após a primeira infestação. Cães dessa raça demonstraram capacidade de desenvolver resistência a R. sanguineus s. l., com diminuição dos parâmetros biológicos e reprodutivos dos carrapatos que se alimentam nestes animais. Foi observado também que cães desta raça reconheciam efetivamente antígenos e respondiam com a produção de anticorpos contra a infestação por carrapatos R. sanguineus s. l.102,103. Apesar de cães beagle serem menos suscetíveis ao parasitismo por R. sanguineus

s. l., e em geral serem encontrados naturalmente com menores cargas parasitarias, estes ainda estão naturalmente infestados5. Ao se comparar o desenvolvimento de R. sanguineus s. l. nas duas raças de cães acima mencionadas, beagle e cocker spaniel inglês, constatou-se um melhor desempenho biológico e reprodutivo de carrapatos recuperados de cães da raça cockers. Nas duas raças, observou-se um efeito na das infestações sucessivas havendo uma diminuição do número de larvas e ninfas ingurgitadas o que ocorreu provavelmente pelo desenvolvimento de uma resposta imune contra estes dois estágios. Porém, a conversão do peso corporal em ovos e a percentagem de ecdise só foram afetadas nos carrapatos alimentados nos cães da raça beagle. Conclui-se que quando comparada as duas raças, cães da raça beagle apresentam uma resposta imune mais efetiva contra o parasitismo deste carrapato, sendo, portanto, um hospedeiro resistente48. Posteriormente foi avaliado o envolvimento da quimiorrecepção na escolha do R. sanguineus s. l. entre as duas raças cocker spaniel inglês (sensível) e beagle (resistente). Avaliou-se a escolha deste carrapato por cães das duas raças em três infestações ambientais e observou-se que, de modo geral, maior número de carrapatos foi observado nos cães da raça Cocker spaniel inglês. Nos testes comportamentais com substâncias colhidas da pele das duas raças, após 1 hora de exposição os carrapatos já se arrestaram sobre as flanelas, sendo significativamente maior número em flanelas de cockers do que de beagles, havendo um aumento no número de carrapatos arrestados sobre ambas as flanelas após 18 e 24 horas, mas as de cockers continha significativamente mais carrapatos arrestados. Os resultados obtidos no teste de olfatometria com as substancias colhidas das duas raças, indicaram uma repelência para os beagles18. Borges et al.19 observaram que cães beagle possuem cerca de 150 vezes mais compostos voláteis em seu perfil cromatográfico que cães cocker spaniel inglês. De acordo com Pickett et al.20 animais não-hospedeiros emitem uma serie de compostos voláteis que são utilizados em sua defesa, assim como alguns animais de espécies hospedeiras que podem ter essa capacidade devida a certas condições. Esses compostos voláteis podem ser utilizados para manipular o comportamento de artrópodes parasitos. No odor dos beagles foram identificados compostos exclusivos e alguns que estavam em maior quantidade tais como 2- hexanona, benzaldeido, nonano, decano e undecano. Estes cinco compostos foram avaliados em ensaios de olfatometria e em placa de Petri e foi observado que a mistura de 2-hexanona e benzaldeido, causou repelência estatisticamente similar ao DEET. Desta forma conclui-se

que estes compostos permitem ao R. sanguineus s. l. identificar e evitar parasitar os cães resistentes da raça beagle19.

2.6. Uso de compostos voláteis de não-hospedeiros (non-host) para o controle de artrópodes

Apesar da manipulação de artrópodes vetores com compostos derivados de espécies não-hospedeiras e hospedeiros resistentes, ter um poder de adaptação maior visto seu espectro espécie-especifico em geral20,21, os dados de seu uso efetivo no controle ou manipulação destes artrópodes ainda são escassos. Um dos produtos à base destes compostos já em via de comercialização é o No Reds®, este produto conta com compostos como bis (2-etil-hexil) adipato e 2,2,4 trimetil 1,3 -pentanodiol e diisobutirato presentes na secreção da glândula uropigial de patos que em geral não são parasitados por ácaros hematófagos como Dermanyssus galinae e Ornithonyssus bursa. A mistura destes compostos em testes in vitro em peles de galinha causou significante redução da alimentação durante o período de teste na pele das galinhas. Ainda durante os testes in vivo o composto demonstrou eficácia ao reduzir a mortalidade em lotes de pintos submetidos a infestações. Além disso, verifica-se que quando a ave ingere estes compostos em associação com ração as substâncias são absorvidas na digestão e secretadas na pele dissuadindo os ácaros do repasto sanguíneo sobre estes animais23. Em outros modelos parasito e hospedeiro resistentes, a presença de compostos em não-hospdeiros atuando como repelentes já fora indetificados e testados25. Na África Subsaariana, a mosca tsetsé (Glossina spp) é um dos vetores cíclicos da tripanossomíase, uma zoonose de alta prevalência no continente. Compostos identificados de seus hospedeiros preferenciais búfalos (Syncerus caffer) e bovinos (B. indicus) diferem quimicamente e estruturalmente de compostos identificados no antílope (Kobus defassa), um hospedeiro não-preferencial para esta mosca, sendo grande parte dos compostos de não-hospedeiro formados por cetonas, fenóis e ácidos graxos de cadeia longas104. Ainda em ensaios eletrofisiológicos estes compostos demonstram atividade fisiológica para mosca tsetsé104. Sendo que a mistura de compostos que demonstram atividade eletrofisiológica δ- octalactone, carvacrol, e C8-C13 metil cetonas (6-metil-5-hepten-2-ona, 2-octanona, 2- nonanona, 2-decanona, 2-undecanona, 2-dodecanona, 6,10-dimetil-5,9-undecadien-2-ona) quando apresentados em túnel de vento para as moscas, foi capaz de inibir a resposta de voo destas, fazendo com diminuíssem tempo e distância e retornando a sua fonte original105.

Posteriormente os compostos identificados no odor do não hospedeiro K. defasa foram misturados em classe por cetonas, ácidos carboxílicos, fenóis, além de mistura entre classes de compostos, para verificar a redução da captura de Glossina spp. em armadilhas NG2G enriquecidas com urina de bovinos. Cada uma das misturas demonstrou eficácia na diminuição da captura de moscas, sendo que a mistura total, entre todos os componentes, reduziu a captura em até 85%. Sendo ainda realizado um ensaio com apenas cinco compostos (δ-octalactona, guaiacol, geranilacetona, ácido hexanóico e ácido pentanoico), tal mistura foi selecionada baseada em dados obtidos de misturas testadas com as classes de compostos onde se subtraiu sem um dos compostos, ensaios subtrativos. Essa mistura de cinco compostos demonstrou uma redução substancial na captura de moscas em 84%, semelhante a apresentada pela mistura total. Em outro experimento foram colocadas as mesmas armadilhas dentro e fora de um cercado que continha um hospedeiro preferencial das moscas, bovinos, os animais foram amarrados em um suporte e no grupo tratado foram colocados sachês com a mistura total ou os cinco compostos (δ-octalactona, guaiacol, geranilacetona, ácido hexanóico e ácido pentanóico) repelentes durante três horas. A eficácia do teste foi baseado na observação de presença ou ausência de sangue no abdômen de moscas capturadas. Sendo observado uma redução de 94% e 96% da alimentação de moscas sobre os bovinos, que estavam com a mistura total ou a mistura de cinco compostos, respectivamente24. Os repelentes encontrados no odor do antílope (K. defasa) guaiacol, geranilacetona, ácido pentanóico e δ-octalactona, foram testados para a proteção contra o parasitismo de moscas tse tse (Glossina spp.) em bovinos no Condado de Kwale na área costeira do Quênia, a área foi escolhida devido alta taxa de prevalência de tripanossomíase. Foram selecionados e tratados 1.100 bovinos com colares contendo os repelentes durante 24 meses. Este colar trata-se de um sistema individual para bovinos, em forma de arco feitos de aço inoxidável com um reservatório para o repelente, no qual o repelente (mistura era injetada), com um sistema de dispersão, os compostos eram injetados uma vez por mês no volume de 10ml sendo ainda utilizada uma proporção de 2: 1: 3: 3 entre eles (guaiacol, geranilacetona, ácido pentanóico e δ-octalactona). Após 24 meses de experimento observou- se que as coleiras forneceram uma proteção substancial aos bovinos da região, visto que houve uma redução de 80% na taxa de tripanossomíase. Além da redução significativa dos níveis de doença, houve aumento do hematócrito e aumento significativo do peso dos animais avaliados, reduzindo o uso de tripanocidas em 60% na área em questão. Além da

saúde dos animais os níveis de terras agricultáveis dessa região melhoraram significativamente, tendo em vista que estes animais são utilizados também como animais de tração25. A identificação destes compostos provindos de animais não hospedeiros, potencialmente pode gerar uma nova classe de repelentes altamente ativos, devido ao seu papel na ecologia dos artrópodes20,21. No entanto o desenvolvimento de tecnologias de repelentes que usam formulações de liberação lenta são tecnicamente desafiadoras20. Portanto o trabalho aqui desenvolvido teve como objetivo, e quantificar os compostos repelentes presentes nos beagles 2-hexanona e benzaldeido, previamente identificados e testados por Borges et al.19, além de avaliar seu potencial uso em formulações de liberação lenta que simulavam a taxa de liberação encontrada nos beagles, no controle de infestações por R. sanguineus s. l. em cocker spaniel inglês.

3 OBJETIVOS

3.1 Objetivo geral

Quantificar compostos repelentes produzidos por hospedeiros resistentes (cães da raça Beagle), e avaliar seu uso no controle de infestações por R. sanguineus s.l. em cães susceptíveis ao parasitismo deste carrapato (Cocker Spaniel Inglês).

3.2 Objetivos específicos

- Quantificar e avaliar a taxa de liberação dos repelentes 2-hexanona e benzaldeido do odor de cães da raça beagle.

- Produzir formulações de liberação lenta contendo os repelentes naturais, 2-hexanona e benzaldeido em diferentes concentrações e proporções.

- Realizar testes de repelência contra R. sanguineus s. l. em cães artificialmente infestados com formulações repelentes a base de 2hexanona e benzaldeído.

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CAPÍTULO 2: Quantification of brown dog tick repellents, 2-hexanone and benzaldehyde, and release from tick-resistant beagles, Canis lupus familiaris

Jaires Gomes de Oliveira Filho a, André Lucio Franceschini Sarria b, Lorena Lopes Ferreira a, John C. Caulfield b, Stephen J. Powers c, John A. Pickett b, Adalberto A. Pérez de León d, Michael A. Birkett b, Lígia Miranda Ferreira Borges e,∗ a Escola de Veterinária e Zootecnia, Universidade Federal de Goiás, Campus II, Samambaia, Cx. Postal 131, Goiânia, Goiás CEP: 74001-970, Brazil b Biological Chemistry and Crop Protection Department, Rothamsted Research, West Common, Harpenden, Hertfordshire AL5 2JQ, United Kingdom c Computational and Systems Biology Department, Rothamsted Research, West Common, Harpenden, Hertfordshire AL5 2JQ, United Kingdom d USDA-ARS, Knipling-Bushland U.S. Livestock Insects Research Laboratory, Veterinary Pest Genomics Center, 2700 Fredericksburg Road, Kerrville, TX 78028, USA e Instituto de Patologia Tropical e Saúde Pública, Universidade Federal de Goiás, Rua 235 s/n, Setor Universitário, Goiânia, Goiás CEP: 74605050, Brazil

Resumo

A repelência do carrapato Rhipicephalus sanguineus sensu lato por cães da raça beagle, é mediada por compostos orgânicos voláteis (COV’s) 2-hexanona e benzaldeído presentes no odor destes cães. Sabe-se que a localização de hospedeiros por ectoparasitos é afetada pela variação desses componentes no odor e suas proporções. O objetivo deste estudo foi quantificar a taxa de liberação e a proporção de 2-hexanona e benzaldeído em beagles. O odor de três beagles foi coletado, durante quatro dias, durante uma semana (dia 0, dia 1, dia 4 e dia 7). Os compostos foram identificados usando cromatografia gasosa de alta resolução acoplada a espectrometria de massa (GC-MS), e padrões autênticos de compostos foram utilizados para gerar curvas de calibração para quantificação. Ambos os compostos foram encontrados em todos os cães em todos os dias. A quantidade de benzaldeído foi sempre superior à de 2-hexanona e, portanto, sua proporção variou em média (ao longo do tempo) sendo 3,148 ± 0,365, 1,902 ± 0,390, 1,670 ± 0,671 ng mL-1 para beagle 1, 2 e 3 , respectivamente. Não houve efeito significativo (p <0,05, F-test) do tempo. A média geral foi de 2,223 ± 0,387 ng mL-1. Estes resultados documentam pela primeira vez a presença de 2-hexanona e benzaldeído em amostras de odor de beagle em um período de 7 dias. Este conhecimento permite o desenvolvimento de repelentes para proteger cães de contra infestações de R. sanguineus s. l.

Palavras-chaves: 2-Hexanona; Benzaldeido; COV’s; Rhipicephalus sanguineus sensu lato; Não-hospedeiros; GC–MS;

Abstract

We have recently shown that repellency of the tick Rhipicephalus sanguineus sensu lato by the tick resistant dog breed, the beagle, is mediated by volatile organic compounds (VOCs)

2-hexanone and benzaldehyde present in beagle odour. Ectoparasite location of animal hosts is affected by variation in these odour components and their ratios. The aim of this study was to quantify the release rate, and the ratio, of 2-hexanone and benzaldehyde from beagles. The odour of three beagles was collected, for four days, over one week (day 0, day 1, day 4 and day 7). The compounds were identified using coupled high-resolution gas chromatography–mass spectrometry (GC–MS), and authentic standards of compounds were used to generate external calibration curves for quantification. Both compounds were found in all dogs on all days. The amount of benzaldehyde was always higher than that of 2- hexanone and so their ratio varied from unity, on average (over time) being 3.128 ± 0.365, 1.902 ± 0.390, 1.670 ± 0.671 ng mL−1 for beagle 1, 2 and 3, respectively. There was no significant (p < 0.05, F-test) effect of time. The overall mean was 2.233 ± 0.387 ng mL−1. These results further previous findings by documenting the presence of 2-hexanone and benzaldehyde in beagle odour samples covering a 7-day period. This knowledge enables development of repellents to protect dogs from R. sanguineus s. l. infestation.

Keywords: 2-Hexanone; Benzaldehyde; VOC’s; Rhipicephalus sanguineus sensu lato; Non-host; GC–MS;

Introduction

Odour chemicals produced by vertebrates can affect the behavior of antagonistic species [1,2]. For the interaction between vertebrate hosts and ectoparasitic ticks (Ixodidae), host location and non-host avoidance are important interspecific olfactory- mediated processes [3–6]. Ticks use chemical cues to find, attach and aggregate on hosts prior to mating, so the discovery of repellents that can disturb these processes is an important step towards developing new control technologies that could also minimize the risk of tick-borne disease transmission. The brown dog tick, Rhipicephalus sanguineus sensu lato, is a major pest affecting public health and also of veterinary importance [7–10]. R. sanguineus s. l. utilizes volatile semiochemicals (naturally occurring behaviour and development-modifying chemical signals) present in breath, skin of its hosts as a mode of host location [11,12]. Louly et al. [13] demonstrated that a lower number of R. sanguineus s. l. ticks were recovered from the beagle breed compared to the English cocker spaniel, and later demonstrated that R. sanguinueus s. l. uses chemoreception when choosing between these breeds. In olfactometer tests comparing the odours from both breeds, ticks were observed to avoid the odour of beagles. This avoidance behavior elicited by resistant beagles was found to be associated with two compounds, 2-hexanone and benzaldehyde, found in their odour, which act as natural tick repellents [12]. When these two compounds were presented together in a 1:1 ratio, the repellency observed was higher than when they were tested alone, and it was

similar to or even higher than the repellency observed using DEET (N,N-diethyl-3- methylbenzamide), a standard repellent. Alternatives to the common use of synthetic acaricides are necessary because of side effects such as environmental pollution and especially the contamination of animal products [14]. There is also the risk of emergence of tick resistance to acaricides [15–18]. A desirable approach is the integration of biological technologies and volatile compounds (VOCs) that alter tick behavior [19]. Borges et al. [12] found compounds in resistant beagles that could be used in such an approach. However, it has been observed in resistant and susceptible hosts that the naturally occurring association of chemical compounds can blind, mask, or expand the perception of other chemical compounds acting as signals to parasites [20–22]. Thus, the combination, or, in mathematical terms, the ratio of compounds in each host can act synergistically or antagonistically against parasites [20,21,23–25]. Therefore, the aim of this study was to analyze and quantify the release rate, and the ratio of 2-hexanone and benzaldehyde odour in beagles.

Material and Methods

Odour collection

Odour components from one male and two female beagle dogs (all 8 years old) were collected by adapting techniques previously described [26]. Briefly, odour components were adsorbed in a mix of activated charcoal (350 mg, 40 m) (Sigma-Aldrich Co., St. Louis, Missouri, USA) and octadecyl bonded silica (450 mg, Bakerbond®, C18, particle size 40 m) (Sigma-Aldrich Co., St. Louis, Missouri, USA) that was packed in a piece of filter paper (9 × 8 cm piece of Whatman no. 1 qualitative) (GE Healthcare UK Ltd., Little Chalfont, UK) stapled securely to produce an adsorbent sachet (4 × 4 cm). Each adsorbent sachet was encased in a folded piece of stainless steel mesh-wire sheet (100 m; 5 × 10 cm) that stapled securely together. A piece of aluminum foil was used to cover each odour trap. Two odour traps were sown with nylon fishing line to the inner side of a strip of elastic material with Velcro at each end. These strips formed collars for the dogs, fitted with the aluminum covered odour traps, and they were kept at −20 ◦C until use. The aluminum was removed from the exposed side of each odour trap before the collar was placed on the dog’s neck. A different odour trap collar was used every time a dog was sampled for 7 h. A total of four

samples were obtained from each dog, at 1, 4, and 7 days after the initial sample was collected on day 0. After each sampling, odour trap collars were stored in a freezer at −20 ◦C until extraction. The adsorbent mix from two sachets in each of the 12 odour trap collars collected was pooled, and transferred to an octadecylsilane (ODS) cartridge (Accu Bond solid-phase extraction) (J&W Scientific Inc., Folsom, California, USA), eluted with 4 mL of dichloromethane (DCM) bi-distillate (Sigma-Aldrich Co., St. Louis, Missouri, USA), concentrated to 150 μL under a gentle stream of clean nitrogen, and the extract bottled in an amber glass ampoule for storage at −20 ◦C.

Gas chromatography (GC) and coupled GC–mass spectrometry (GC–MS) analysis

Extracts (1 μL) were analysed on an Agilent 6890A GC (Agilent Technologies, Santa Clara, California, USA), equipped with a cool column injector, flame ionization detector (FID), and an HP-1 capillary GC column (50 m × 0.32 mm i.d. × 0.52 m film thickness). The oven temperature was maintained at 30 ◦C for 0.1 min, then programmed to increase at 10 ◦C min-1 until 250 ◦C, and then held for 38 min. For compound identification by coupled GC-MS (VG Autospec Ultima, Manchester, UK), an HP-1 capillary GC column (50 m × 0.32 mm i.d. × 0.52 m film thickness), equipped with a cool on-column injector was used. Ionization was accomplished by electron impact at 70 eV, 250 ◦C. The oven temperature was maintained at 30 ◦C for five minutes and then programmed to increase 5 ◦C min-1 until 250 ◦C. The carrier gas was helium. Tentative identification by GC–MS was confirmed by comparing retention indices of peaks with those of authentic standards and by peak enhancement on GC by co-injection with authentic compounds, using an Agilent 6890 N GC with 10 m × 0.32 mm i.d. HP-1 column. The oven temperature was maintained at 30 ◦C for 0.1 min, then programmed to increase at 10 ◦C min-1 until 250 ◦C, and then held for 30 min.

2-hexanone and benzaldehyde quantification

Analytical curves were constructed for determination of the concentration of 2- hexanone and benzaldehyde in the dog odour extracts. Primary stock standard solutions of 2-hexanone (Aldrich Chemical Co., Ltd., Dorset, UK) and benzaldehyde (Sigma-Aldrich Co., Ltd., Steinheim, Germany) at a concentration of 1000 ng mL-1 were prepared using

DCM bi-distillate (Sigma-Aldrich Co., St. Louis, Missouri, USA) as solvent. Stock solutions were diluted serially to reach a concentration of 10 ng mL-1, which was used to produce five different concentrations of 2-hexanone (2.5, 1.5, 1.0, 0.5 and 0.1 ng mL-1) and benzaldehyde (6.0, 5.0, 4.0, 2.5 and 1.0 ng mL-1). Each final concentration was tested in triplicate. One L of each solution was injected and analysed by GC-FID using the method as shown in the previous section. This method was adopted to account for technical variation in the calibration procedure. Goodness of fit of the calibration curves was determined by linear regression analysis with the chromatographic areas (mAU) obtained from the GC being the response variable, y, and the corresponding concentrations injected to secure those areas, of either 2-hexanone or benzaldehyde compounds (ng mL-1), being the explanatory variable, x. Using the range of concentrations of each compound, the linear regression model was y = c + bx, where c is the intercept and b is the slope of the fitted line. The strength of the relationship in each case was judged in terms of the proportion of variation explained, R2, and the significance (p < 0.05) of the F-test from the analysis of variance (ANOVA) accompanying the regression. Evidence of any curvature was assessed by adding a quadratic term in concentration into the linear model and testing the improvement in fit (F-test). The model was fit using ordinary least squares, which provides estimates of the parameters c and b along with standard errors. The linear formulae obtained from this analysis were used to determine the concentration of each compound in each sample, by substituting in the values of areas, y, for the samples, to calculate the corresponding concentrations, x. Ratios were obtained by dividing the quantity of benzaldehyde by 2-hexanone collected every day from each dog. ANOVA was applied to the concentration data for each compound and the ratio, to assess the significance (p < 0.05, F-tests) of overall difference between beagles and between time points, noting the four repeated measures from each beagle. The limit of detection (LOD) and the limit of quantification (LOQ) were determined in accordance with the linear regression, and are expressed as:

LOD = 3.3 × SE(c)/b

LOQ = 10 × SE(c)/b where: SE(c) is the standard error of the intercept and b is the slope of the analytical curve.

The GenStat (17th edition, © VSN International Ltd., Hemel Hempstead, UK) statistical package was used for analysis. No transformation of the data was required, inspection of residuals confirming that the data broadly complied with the assumptions of the analysis.

Results

Quantitative analysis of tick repellent compounds The presence of 2-hexanone and benzaldehyde in beagle odour samples was confirmed by direct comparison of their mass spectra and retention times,. and GC peak enhancement with authentic standards of 2-hexanone (0.033 ng mL-1) and benzaldehyde (0.016 ng mL-1) (Fig. 1) The full set of 12 chromatograms (three dogs at four time points) is given in Fig. 2. The two compounds were quantified (Table 1) by using fitted analytical calibration curves. Table 2 gives details of data for the ratio of the two compounds. Over the concentration range of 0.1–2.5 ng mL-1 for 2-hexanone, and of 1.0–6.0 ng mL-1 for benzaldehyde, there was a linear relationship for the compounds. There was a strong correlation between the concentrations of the compounds in the dilution series of the standard samples with the corresponding peak area in the chromatograms. Table 3 and 4 show the ANOVA output of the linear regression models for 2-hexanone and for benzaldehyde along with the estimated parameters (intercepts and slopes) in the models. For 2-hexanone, there was a highly significant regression (p < 0.001, Ftest) and also for benzaldehyde (p < 0.001, F-test). For the latter compound the intercept value was not statistically different from zero (p > 0.05, F-test) and there was no evidence (p < 0.05, F- test) of curvature for either compound 2-hexanone (p = 0.822, F-test) of for benzaldehyde (p = 0.207, F-test). The adequacy of the linear model was also assessed in terms of the very high proportion of variance explained (R2), being 98.27% for 2-hexanone and 99.48% for benzaldehyde.

Fig. 1. Co-injection of Beagle odour extracts with authentic standards 2-hexanone (0.033 ng mL-1) and benzaldehyde (0.016 ng mL-1); (A): synthetic compounds; (B): Dichloromethane extract from beagle 1 day 0; (C): Co-injection of beagle sample with synthetic 2-hexanone and benzaldehyde. The peak for 2-hexanone is labelled as 1; the peak for benzaldehyde is labelled as 2.

Fig. 2. The GC and GC/MS of DCM extracts of the odours of the three beagles, in all four days evaluated. The 12 spectra were highly similar, and 2-hexanone and benzaldehyde were present in all extracts. A1: Beagle 1 day 0; A2: Beagle 1 day 1; A3: Beagle day 4; A4: Beagle day 7; B1: Beagle 2 day 0; B2: Beagle 2 day 1; B3: Beagle 2 day 4; B4: Beagle 2 day 7; C1:

Beagle 3 day 0; C2: Beagle 3 day 1; C3: Beagle 3 day 4; C4: Beagle 3 day 7; the peak for 2- hexanone is indicated labelled as 1; and the peak for Benzaldehyde is labelled as 2

Table 1. Concentration of 2-hexanone and benzaldehyde in odour samples of three beagles across four days during one week, having analysed samples by GC and conversion of peak areas from the spectra into concentrations (ng mL−1) via linear regression models (see Table 3 and Table 4). Beagles 1 and 3 are female, beagle 2 is male. There were no significant (p < 0.05, F-tests) effects of time or between beagles. 2-Hexanone Benzaldehyde Beagle Day Day Day Day Day Day Day Day 0 1 4 7 Mean SE 0 1 4 7 Mean SE 1 0.652 0.717 0.662 0.633 0.666 0.021 2.407 2.278 1.479 2.157 2.080 0.239 2 1.586 1.150 2.157 1.175 1.517 0.272 2.364 1.480 6.038 2.386 3.067 1.169 3 1.329 1.919 1.305 3.405 1.989 0.569 1.961 3.545 2.900 3.865 3.068 0.485 Mean 1.189 1.262 1.374 1.738 1.391 2.244 2.434 3.472 2.802 2.738 SE 0.227 0.287 0.354 0.693 0.269 0.116 0.491 1.100 0.437 0.421

Table 2. Ratio of benzaldehyde to 2-hexanone in odour samples of three beagles across four days during one week. Beagles 1 and 3 are female, beagle 2 is male. There was some evidence of real difference between beagles (p = 0.057, F-test), the standard error of the difference between the means for the beagles was 0.506 on 6° of freedom. Beagle. Day 0 Day 1 Day 4 Day 7 Mean SE 1 3.691 3.176 2.235 3.410 3.128 0.365 2 1.491 1.286 2.800 2.030 1.902 0.390 3 1.475 1.848 2.223 1.135 1.670 0.271 Mean 2.219 2.103 2.419 2.192 2.233 SE 0.736 0.560 0.190 0.662 0.287

Table 3. Summary of the output of the ANOVA for the linear regression analysis of 2-hexanone. The degrees of freedom (df), sums of squares (SS), mean squares (MS), variance ratio (VR) and p-value for the F-test of the variance ratio (F.pr.) are given along with the estimated coefficients in the linear regression model, their standard errors (SE), partial t-statistics on 13 df and p-values for the t-tests of these t-statistics. df SS MS VR F.pr. Regression 1 3874.42 3874.42 2530.14 <0.001 Residual 13 19.91 1.531 Total 14 3894.33 278.17 Percentage variance accounted for (R2) 99.4

df SS MS VR F.pr. Standard error of observations is estimated to be 1.24 Coefficients SE t (13) T pr. Constant (c) 0.289 0.534 0.54 0.598 Concentration (b) 19.242 0.383 50.30 <0.001

Table 4. Summary of the output of the ANOVA for the linear regression analysis of benzaldehyde. The degrees of freedom (df), sums of squares (SS), mean squares (MS), variance ratio (VR) and p-value for the F-test of the variance ratio (F.pr.) are given along with the estimated coefficients in the linear regression model, their standard errors (SE), partial t-statistics on 13 df and p-values for the t-tests of these t-statistics. df SS MS VR F.pr. Regression 1 5481.96 5484.963 2508.86 <0.001 Residual 13 28.41 2.185 Total 14 5510.37 393.598 Percentage variance accounted for (R2) 99.4 Standard error of observations is estimated to be 1.48 Coefficients SE t (13) T pr. Constant (c) −0.981 0.881 −1.11 0.286 Concentration 10.754 0.215 50.09 <0.001 (b)

Estimation of the quantity of 2-hexanone and benzaldehyde produced by the beagles

The range in concentration of 2-hexanone in the odour from beagles was 0.633 ng mL-1 in beagle 1 on the 7th day to 3.405 ng mL-1 in beagle 3 on the 7th day (Table 1). For benzaldehyde, the range observed was 1.479 ng mL-1 in beagle 1 on the 4th day to 6.038 ng mL-1 in beagle 2 on the 4th day. The ratio of benzaldehyde:2-hexanone varied from 1.135 to 3.691, being, on average (over time), 3.128 ± 0.365, 1.902 ± 0.390, 1.670 ± 0.671 ng mL- 1 for beagle 1, 2 and 3, respectively. The overall average was 2.233 ± 0.387 ng mL-1. ANOVA revealed no significant effect of time points (p < 0.05, F-tests) for either of the compounds or the ratio. There was some evidence of real difference (p = 0.057, F-test) between beagles for the ratio, this evidence clearly being derived from the effect of beagle for 2-hexanone (p = 0.096, F-test) rather than benzaldehyde (p = 0.563, F-test). Beagle 1 had a far greater ratio than the other two beagles (Table 2), due to much lower mean concentrations of 2-hexanone over time (Table 1). For 2-hexanone the LOD and the LOQ

were found to be 0.092 and 0.278 ng mL-1 respectively and for benzaldehyde the LOD and the LOQ were found to be 0.270 and 0.819 ng mL-1 respectively. For benzaldehyde the LOQ was smaller than the first point of the calibration curve (≤1.00 ng mL-1) indicating that, assuming linearity, smaller quantities than the smallest calibrated could be estimated from corresponding chromatographic areas, although for robust estimates from the regression analysis it would still be advisable to remain within the limits of the calibration.

Discussion

Here, we report on validation of an analytical method to characterize VOCs that have a repellent effect, and quantify the relatively minor amounts of the tick repellents 2- hexanone and benzaldehyde in the odour of beagles in a consistent and reliable manner and hence also the natural ratio of benzaldehyde to 2-hexanone in the odour. Our results enhance previous findings [12] by documenting the presence of 2-hexanone and benzaldehyde in beagle odour samples covering a 7-day period. Previous studies showed that the beagle is resistant to tick infestation and that repellency is associated with host odour components [12,27]. Although the 1:1 combination of 2-hexanone and benzaldehyde was shown to be as repellent as DEET, the in vivo variation in their concentrations over time was not documented. The actual benzaldehyde:2-hexanone ratio, based on quantitative readings in the current study, ranged from 1.1:1 to 3.7:1, average 2.2:1, which suggests there is most usually around 2-fold greater benzaldehyde than 2-hexanone in the natural repellent. To our knowledge, this is the first study that quantifies these repellent compounds, produced naturally by animals, for a period longer than one day in order to document their persistence in proportions shown to repel ticks in vivo and in vitro. A 2:1 benzaldehyde:2-hexanone ratio should be tested in future experiments. Odour compounds can be specific to particular animals and function as chemical signatures from an ecological perspective [12,26,28,30,32]. VOCs acquired and developed during life are influenced by the physiological state of living organisms [30,31,33–36]. Plant VOCs can change daily and weekly [25,37]. Levels of the tick repellents (4aS,7S,7aR-) and (4aS,7S,7aS)-nepetalactone in the essential oil of Nepeta cataria (Lamiaceae) vary during the course of ten weeks [38]; however, the quantitative and proportional fluctuation of the compounds across time was not reflected in variation of efficacy. Remarkably, the analytical results for 2-hexanone and benzaldehyde reported here and obtained from older beagles are

comparable to those reported in younger dogs [12]. An important branch of chemical ecology research is the innovation and adaptation of technologies to determine the ratio of repellent compounds in biological matrices. Parasitism perhaps drives the evolution of repellent mixtures produced by hosts, and the actual mixture composition and proportion of its components may be linked to host susceptibility or resistance [20,21,23–25]. Knowledge of the ratio of principal bioactive components in natural mixtures with repellent effects also facilitates the development of formulations to protect humans or domestic animals. As previously done by others documenting the importance of elucidating the ratio of repellents in natural substances to inform formulation [25,29], we have shown that benzaldehyde is relatively more abundant than 2-hexanone in beagle odour and suggest that a 2:1 benzaldehyde:2-hexanone ratio should be used to develop and test sustained release formulations. The cosmopolitan brown dog tick, R. sanguinueus s. l., is one of the species making the global problem of ticks and tick-borne diseases more complex [18,39,40]. Developing strategies that integrate diverse technologies is an approach that will help manage ticks, and the pathogens they transmit, in a sustainable manner [41,42]. The use of anti-tick vaccines has been proposed as a way to protect dogs from infestation and tick-borne disease transmission [42,43]. Our findings provide additional supportive information for the development of a formulation combining benzaldehyde and 2-hexanone to protect dogs susceptible to infestation with R. sanguinueus s. l. [12]. Integrated treatment, in areas where there are tick infestations, that combines anti-tick vaccination with the use of a formulation based on repellents discovered in beagle dogs would protect not only dogs, but also humans from tick parasitism and tick-borne zoonotic pathogens.

Acknowledgments

We thank the National Council of Technological and Scientific Development (CNPq) (Grant no. #/2013-9), Brazil, for its financial support of this project and for supply a Scholarship to LMFB (#/2015-0). Rothamsted Research receives grant-aided support from the Biotechnology and Biological Sciences Research Council (BBSRC) of the United Kingdom. USDA is an equal opportunity provider and employer.

References

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CAPÍTULO 3: Brown dog tick, Rhipicephalus sanguineus sensu lato, infestation of susceptible dog hosts is reduced by slow release of semiochemicals from a less susceptible host

Jaires Gomes de Oliveira Filho a,1, Lorena Lopes Ferreira a,1, André Lucio Franceschini Sarria b, John A. Pickett b, Michael A. Birkett b, Gabriel Moura Mascarin c, Adalberto A. Pérez de León d, Lígia Miranda Ferreira Borges e,∗ a Escola de Veterinária e Zootecnia, Universidade Federal de Goiás. Campus Samambaia, Avenida Esperanc ¸ a, s/n, Campus Universitário, CEP: 74690-900, Goiânia, Goiás, Brazil b Biological Chemistry and Crop Protection Department, Rothamsted Research, West Common, Harpenden, Hertfordshire, AL5 2JQ, United Kingdom c EMBRAPA Arroz e Feijão, Rodovia GO-462, Km 12, Zona Rural, Caixa Postal: 179, CEP: 75375-000, Santo Antônio de Goiás, Goiás, Brazil d USDA, ARS, Knipling-Bushland U.S. Livestock Insects Research Laboratory and Veterinary Pest Genomics Center, 2700 Fredericksburg Road, Kerrville, TX, 78028, USA e Instituto de Patologia Tropical e Saúde Pública. Universidade Federal de Goiás, Rua 235s/n, Setor Universitário, CEP: 74605050, Goiânia, Goiás, Brazil

Resumo Cães domésticos são hospedeiros comuns para o ‘carrapato marrom do cachorro’, Rhipicephalus sanguineus sensu lato, mas os níveis de infestação variam entre as raças. Beagles são menos suscetíveis a infestações de carrapatos do que cocker spaniels ingleses devido à produção de 2-hexanona e benzaldeído que atuam como repelentes voláteis. Aqui relatamos o uso de um protótipo de formulações de liberação lenta desses compostos para reduzir a infestação de R. sanguineus s. l. em cães cocker spaniel ingleses. Doze cachorros foram distribuídos aleatoriamente em dois grupos com seis cães cada. O grupo tratado recebeu coleiras com formulações de liberação lenta dos compostos, enquanto o grupo controle recebeu coleiras com sem formulações. Foram realizadas cinco infestações ambientais, com todos os estágios do carrapato, os cães foram contados duas vezes ao dia durante 45 dias. A contagem do número de estágios encontrados por cão foi ajustada individualmente a modelos de efeitos mistos lineares com medidas repetidas e distribuição normal de erros. A infestação média do carrapato no grupo tratado foi significativamente menor do que no grupo controle. Para larvas e ninfas, observou-se uma diminuição na infestação de carrapatos na quinta contagem, e para adultos, menores contagens médias foram observadas em todas as contagens durante o tratamento. Os compostos não interferiram na distribuição dos carrapatos no corpo dos cachorros, pois uma porcentagem semelhante de carrapatos foi encontrada na metade anterior dos cães (54,5% para o grupo controle e 56,2% para o grupo tratado). Os parâmetros biológicos e reprodutivos dos carrapatos não foram afetados pelos repelentes. Este estudo destaca pela primeira vez o uso potencial de uma nova formulação baseada em alomônio (repelente) para redução da infestação de carrapatos em cães suscetíveis.

Palavras-chaves: não-hospedeiros; Alomonios; 2-Hexanona; Benzaldeido; Formulação; PML’s. Abstract Domestic dog breeds are hosts for the brown dog tick, Rhipicephalus sanguineus sensu lato, but infestation levels vary among breeds. Beagles are less susceptible to tick infestations than English cocker spaniels due to enhanced production of 2-hexanone and benzaldehyde that act as volatile tick repellents. We report the use of prototype slow-release formulations of these compounds to reduce the burden of R. sanguineus s. l. on English cocker spaniel dogs. Twelve dogs were randomly assigned to two groups with six dogs each. The treated group received collars with slow-release formulations of the compounds attached, while the control group received collars with clean formulations attached. Five environmental infestations were performed, with the number of ticks (at all stages) on the dogs being counted twice a day for 45 days. The counts on the number of tick stages found per dog were individually fitted to linear mixed effects models with repeated measures and normal distribution for errors. The mean tick infestation in the treated group was significantly lower than in the control group. For larvae and nymphs, a decrease in tick infestation was observed at the fifth count, and for adults, lower average counts were observed in all counts. The compounds did not interfere with the distribution of the ticks on the body of the dogs, as a similar percentage of ticks was found on the anterior half of the dogs (54.5% for the control group and 56.2% for the treated group). The biological and reproductive parameters of the ticks were not affected by the repellents. This study highlights for the first time the potential use of a novel allomone (repellent)-based formulation for reduction of tick infestation on susceptible dogs.

Keywords: Non-host; Allomone; 2-Hexanone; Benzaldehyde; Formulation; SLM’s;

Introduction The brown dog tick, Rhipicephalus sanguineus sensu lato, has a cosmopolitan host distribution and, in addition to parasitizing domestic dogs, can parasitize birds, livestock and human beings (Borges and Silva, 1994; Louly et al., 2006; Rodríguez-Vivas et al., 2016; Szabó et al., 2012). R. sanguineus s.l. comprises a species complex (Nava et al., 2015; Szabó et al., 2005), and is of veterinary and public health importance due to its obligate blood feeding habit and role as a vector of pathogens such as Babesia vogeli, Ehrlichia canis, Anaplasma platys, Rickettsia rickettsii and R. conorii (Cardoso et al., 2010; Eremeeva et al., 2011; Pacheco et al., 2011; Socolovicvhi et al., 2009). Tick infestation levels can vary within a single host species according to host breed, age, immunological state, and individual semiochemical production (Bunnell et al., 2011; Pickett et al., 2010; Weldon, 2010). Our hypothesis, relating to the ecological basis of ectoparasite repellency in hosts (Pickett et al., 2010), is that individual hosts, either closely related taxonomically or from a single taxon, differ in their interaction with ectoparasites

due to differences in the production and emission of non-host chemical signaling. Furthermore, individual signal components emitted by each host can act synergistically or can exert antagonism to other ectoparasites (Nielsen et al., 2015; Weldon, 2010). For example, a blend of volatiles produced by the non-host waterbuck, Kobus defassa, was identified as being repellent to tsetse flies, Glossina morsitans, (Bett et al., 2015; Gikonyo et al., 2002, 2003). However, little is known about host natural odors that are repellent for ticks. In our previous work where it was shown that beagles were less infested with R. sanguineus s. l. than the English cocker spaniel, it was suggested that a host factor could determine differential tick load (Louly et al., 2009, 2010). Our subsequent work identified two volatile small lipophilic molecules (SLMs), 2-hexanone and benzaldehyde, which are produced in greater amounts in beagles, and which act as natural repellents against R. sanguineus s. l. (Borges et al., 2015), and demonstrated constitutive release of these two SLMs in the odor of beagles (Oliveira Filho et al., 2016). Acaricidal treatments remain the most common practice to control R. sanguineus s. l. However, the indiscriminate use of products containing synthetic acaricides represents a strong selective pressure that results in the emergence of resistant tick populations to those active ingredients (Borges et al., 2007; Eiden et al., 2015; Miller et al., 2001). Considering the role of non-host chemical signaling in host selection by ectoparasites, it has been suggested that such signaling, produced by resistant hosts, may form the basis of ecologically-based repellents to control ectoparasite infestation (Pickett et al., 2010; Weldon, 2010), and may also reduce the risk of exposure to vector-borne pathogens. Following our earlier work on biting fly repellents from non-preferred hosts (Birkett et al., 2004; Logan et al., 2008, 2009), we hypothesized that tick numbers on a susceptible animal host could be reduced by application of natural repellents discovered in the odor of less preferred conspecifics. Here, we investigate the effect of a prototype system, delivering 2-hexanone and benzaldehyde via slow release from polyethylene sachets, upon R. sanguineus s. l. infestation on English cocker spaniels, following artificial exposure under environmentally controlled conditions.

Material and Methods Animals The use of animals (dogs and rabbits) in this study was approved by the Committee on Ethical Animal Use of the Federal University of Goiás (CEUA/UFG, protocol number 024/2014). The care and use of the animals during this study were undertaken according to bioethics and animal welfare guidelines required by CEUA/UFG. Eight male and four female English cocker spaniel dogs, with ages varying from 50 to 116 days old, were obtained from different breeders. They were treated for intestinal worms (Drontal Puppy – Bayer®), received all applicable vaccinations (Vanguard Plus and Defensor – Pfizer®), and were not treated with acaricides for 30 days before the study was initiated. All the dogs had their pelage cut to the same length prior to the initiation of the study, in order to offer the same conditions for tick infestation and also to facilitate tick counts. Dogs were housed in a kennel at the Veterinary and Animal Husbandry School of the Federal University of Goiás, Brazil. The kennel has a total area of 26.6 m2, with an internal area of 9.8 m2 that was used to inspect the dogs and six individual stalls each one with 2.4 m2 being a cover area with 1.28 m2 and an open area with 1.12 m2. The floor is cemented and the walls of the kennel are glazed for easy cleaning. A week prior to the beginning of the experiment, all areas of the kennel were sanitized with a flamer three times in a week. During the progress of the experiment, the animals were fed twice a day (Golden Filhotes-Premier®), following the amounts recommended by the manufacturer and given water ad libitum, while the floor was cleaned daily with water and neutral detergent. The health of the animals was monitored daily and clinical pathology exams were conducted when necessary. Dogs were randomly divided composing two groups (treated and control) with six animals each (four males and two females per group). The dogs were randomly divided into three per stalls in order to facilitate the handling of animals and cleaning facilities.

Ticks Engorged females of R. sanguineus s.l. were harvested from naturally infested dogs for the municipality of Goiânia, Goiás, Brazil, the establishment of a colony. The ticks were maintained in an acclimatized chamber (27 ± 1 ◦C and 80% R.H.) and fed on rabbits (Oryctolagus cuniculus) to obtain differing life stages for use in dog infestations (Louly et al., 2010). The ticks used in the experiments were aged between one and three weeks old. Rabbits were removed from use after two consecutive infestations. During infestations, rabbits were examined daily and none showed symptoms of damage due to tick parasitism.

Preparation and use of prototype non-host semiochemical delivery system Samples of analytical grade 2-hexanone (Aldrich Chemical Co. Ltd., Dorset, UK) and benzaldehyde (Sigma-Aldrich Co. Ltd., Steinheim, Germany) were used in this study. Sheets of cellulose sponge (0.5 cm thick, code 0032 6865 J. Sainsbury plc), previously soaked in chloroform overnight then washed in chloroform 3 times before drying in a fume cupboard, were cut into pieces (∼2.5 × 2 cm) and either treated with 400 L of benzaldehyde or 2-hexanone, or left untreated. Polyethylene sachets were prepared by heat sealing the sponges inside polyethylene sheeting (∼3 × 3 cm, Al Packaging Ltd., London film type LFT size 50 mm gauge and 62.5 mic lift). The mean release rate per day of the compounds from the sachet formulations was determined over several weeks using dynamic headspace collection (air entrainment) and GC analysis. Thus, three sachets filled with either benzaldehyde or 2-hexanone were enclosed in a glass vessel (700 mL). Air was pumped through an activated charcoal filter into the vessel (1 L/min) and was then drawn (500 cc/min) into tubes containing the adsorbent Porapak Q (50 mg). After one hour, volatiles collected on the Porapak Q were eluted with 750 L of redistilled diethyl ether and the samples were stored at -20 ◦C until analysis. Extracts (1 L) were analysed on an Agilent 6890 N GC fitted with a 10 m × 0.32 mm i.d. HP-1 column. The oven temperature was maintained at 30 ◦C for 0.1 min, then programmed at 10 ◦C min-1 to 250 ◦C and held for 30 min. The quantity of compound captured per sample was expressed in g/day using the analytical method described by Oliveira Filho et al. (2016). For 15 days, the release rates were 30.44 ± 1.12 g/day for benzaldehyde and 14.38 ± 1.26 g/day for 2-hexanone, decreasing sharply to 11.18 g/day and 1.29 g/day, respectively, after the 15th day. The ratio of released 2-hexanone and benzaldehyde broadly matched the ratio of naturally release from beagles (Oliveira Filho et al., 2016), and so experiments were conducted with these prototype formulations. The sachets were stored at -20 ◦C until required for tests. Sachets were attached to dog collars using a stapler shooter containing light duty staples (5/16” 8 mm – Stanley) and covered with gauze to prevent injury to the dogs’ necks. A single 2-hexanone sachet and a single benzaldehyde sachet was attached per dog collar. A collar with one untreated sachet was used in each of the untreated control group dogs.

Tick infestation, evaluation, and sampling

Five artificial environmental infestations were established each time by positioning breeding vials against the stall walls approximately 80 cm above the floor to release 2000 larvae, 100 nymphs, and 60 adults (30 males and 30 females) per stall. The first infestation was held 10 days before the attachment of the first collar, and afterwards four more infestations were carried out, every seven days. After the first infestation dogs were shaved. The collars were changed after two weeks, and were removed one week after the fifth infestation and a count followed by an additional week. The dogs were rotated among the stalls on a weekly basis and in a clockwise fashion, which promoted standard experimental conditions to the animals. Dogs were thoroughly inspected every day for larvae, nymphs and adults in the morning and afternoon, starting two days after the initial infestation and until seven days after the second set of repellent delivery collars were removed. A map of 24 body areas, including R. sanguineus s. l. sites, was used to standardize tick counts (Otranto et al., 2005). The final number of ticks per day was determined by the maximum number of ticks per area found in one day, and the counts representing the total of ticks found per dogs during a week, except at the first count that lasted 10 days. During the counts, the dogs were brushed twice a day to collect all engorged stages of R. sanguineus s. l. Male ticks were not removed during the brushes. The ticks collected from the dogs were placed in a climate chamber (27 ± 1 ◦C and 80% R.H.) to assess the engorged female weight (EFW), egg mass weight (EMW), conversion of body weight on eggs (CBWE), conversion of body weight on larvae (CBWL), percentage of larval- hatchability (LH), percentage of larval ecdysis (LE), and percentage of nymphal ecdysis (NE). EFW was obtained after manual detachment of females while EMW was determined 15 days after the onset of egg laying. LH was evaluated by averaging the counts of two evaluators according to the estimate of the percentage of larvae that hatched from eggs (Bechara et al., 1994).

Statistical analysis Count data on the number of larvae, nymphs and adults found per dog were individually fitted to linear mixed effects models with repeated measures and normal (Gaussian) distribution for errors. Normality assumptions were previously checked based on Shapiro-Wilk and Bartlett tests. In addition, goodness-of-fit of the models were assessed using residual plots. These models were implemented using the “lme4” package (Bates et

al., 2015, https:// CRAN.R-project.org/package=lme4) from the free statistical software R (R Core Team, 2014; http://www.R-project.org/.). The linear predictors were represented by treatment (control vs. treated dogs wearing the repellent collar), infestations, and their interaction term. Since all dogs were repeatedly evaluated for all four weeks, dog was included as a random effect in models (i.e., to allow for non-independence of each time measurement from a given dog). Therefore, the model may be written as:

Yijn = β0 + Xi + Tj + X ∗ Tij+n+εij

Where Y ijn is the value of the outcome variable (number of larvae, nymphs or adults per dog) for the i-th treatment of the j-th infestation at the n-th replicate (i.e., dog), ˇ0 is the intercept, Xi is the treatment effect, Tj is the infestation time, X*Tij is the interaction term, n is the random effect for dogs, and εij is the error for treatment i in time j. Then, type 3 F-tests with Satterth- waite’s approximation for degrees of freedom was employed to assess significance (P-values) of fixed effects using the “lmerTest” package from R (Kuznetsova et al., 2015, http://CRAN.R-project. org/package=lmerTest). When the interaction term was significant, multiple pairwise comparisons were performed based on differences of least squares means at P < 0.05. The number of engorged larvae, nymphs and females recovered during the brushes was insufficient to evaluate the biological and reproductive parameters over the successive infestations. The Student t test at P < 0.05 was used to compare these parameters between treated and control groups adding the ticks recovered from all infestations, during the time the dogs were wearing the repellent collars.

Results

During the first infestation of R. sanguineus s.l. on dogs, conducted before the attachment of repellent-treated collars, the number of ticks (larvae, nymphs and adults) in the two groups was virtually similar, ranging from a rate of 30 ticks found on treated group and 27 ticks on the control group (larvae: t = 0.2774, df = 5, P = 0.7926; nymphs: t = 0.0, df = 5, P = 1.0; adults: t = 0.3974, df = 5, P = 0.7075). In the last count (6th week) realized without artificial infestation and in the absence of collars, a total (larvae, nymphs and adults) of 203 and 204 ticks in treated and control groups was observed, respectively. No larvae were found in either group, only five nymphs were found in the treated group vs. six in

control group (t = 0.3492, df = 5, P = 0.7412), and the average number of adults was 198 for untreated dogs vs. 198 for treated dogs (t = 0.1661, df = 5, P = 0.8746). According to the F-test, treatment and infestation time (or sampling date) had a significant effect on the number of larvae, nymphs and adults of R. sanguineus s. l. found on dogs, as their interaction term was significant (P = 0.0110, P = 0.0430, P = 0.0373, respectively). This indicates that all life stages of this tick species considerably decreased across time in the presence of the repellent formulation (Fig. 1). The compounds did not interfere with the distribution of the ticks on the body of the dogs, as a similar percentage of ticks was found on the anterior half of the dogs (54.5% for the control group and 56.2% for the treated group). A more pronounced reduction in tick infestation due to the repellent effect was noted for larvae at the 5th count, with averages of 4.67 larvae in the treated group against 10.67 larvae in the control group (P < 0.0001). Likewise, the counts of nymphs showed a significant difference in the same interval with average loads of 3.33 and 10.67 nymphs in the control and treated groups respectively (P < 0.0001). Averages for adult counts always showed significant reductions in infestation upon artificial infestations in the four consecutive weeks for animals wearing the repellent collar compared to those wearing untreated collars. For instance, in the 2nd infestation, the means of adult ticks found were 12.67 and 21.33 in treated and control groups respectively (P = 0.002). In the 3rd infestation, the mean was 34.5 in treated group vs. 39.67 in control group (P = 0.006), in the 4th infestation the number was 41 in treated and 49.67 in control group (P < 0.0001), and in 5th infestation the mean of adults reached 39.67 and 48 in treated and control, respectively (P < 0.0001) (Fig. 1). Regardless of the treatment, all tick life stages tended to increase with time, and population peaks were noted at the 4th or 5th week of evaluation (Fig. 1). Overall, tick infestation loads in dogs wearing the collar containing the repellent formulation exhibited significantly lower numbers of larvae (P = 0.00049), nymphs (P = 0.00027) and adults (P < 0.0001), when compared to untreated dogs from the control group (Fig. 2). The weight of the female ticks, egg conversion, larval hatchability and ecdysis were statistically similar in treated and control groups (Table 1), suggesting that the repellent formulations had no effect on the biological parameters of this tick species.

Fig. 1. Mean (±SE, standard error) number of ticks (larvae, nymphs, and adults) retrieved from untreated (control) and treated (repellent collar) dogs across infestation periods. Vertical arrows indicate when dogs received the repellent collar. At 4th infestation interval, the repellent collar was replaced by a new one and in the 5th the repellent collar was removed. The counts during a week, except the first count during 10 days. Significant difference between control and treated dogs is indicated by P < 0.05 (*) or P < 0.01 (**).

Fig. 2. Overall mean (±SE, standard error) number of ticks (larvae, nymphs, and adults) retrieved from untreated (control) and treated (repellent collar) dogs. Significant difference between control and treated dogs is indicated by P < 0.05 (*) or P < 0.01 (**).

Table 1. Mean (±SD) of biological parameters of Rhipicephalus sanguineus sensu lato ticks obtained along successive infestations on dogs treated or non- treated with 2-hexanone and benzaldehyde. Groups Parameters Treated Control EFW (g) 93.00 ± 30.00a(n = 10) 81.00 ± 34.00a(n = 10) EMW (g) 50.00 ± 25.00a(n = 10) 40.00 ± 21.00a(n = 10) LH (%) 83.55 ± 31.64a 83.33 ± 31.72a

Groups Parameters Treated Control CBWE (%) 51.99 ± 16.41a 52.61 ± 20.72a CBWL (%) 42.87 ± 22.39a 49.45 ± 19.96a LE (%) 64.08 ± 21.27 a (n = 23) 79.16 ± 24.57 a(n = 39) NE (%) 77.77 ± 40.36 a(n = 9) 75.17 ± 41.83 a(n = 7) All parameters evaluated were statistically similar between treated and control groups by t- Student test (P > 0.05). EFW: engorged female weight, EMW: egg mass weight, LH: larval- hatchability rate, CBWE: conversion body weight on eggs, CBWL: conversion body weight on larvae, LE: percentage of larval ecdysis, NE: percentage of nymphal ecdysis.

Discussion

The results presented here support the hypothesis that non-host unsuitability can be conferred to a suitable host by treatment with non-host semiochemicals (Borges et al., 2015; Pickett et al., 2010; Weldon, 2010; Weldon, 2013). Our findings document, for the first time, in vivo activity of a natural repellent blend containing odor components of a resistant host, which could be regarded in general as a safer tool to be used to manage R. sanguineus s. l. infestations on dogs, and perhaps other susceptible host species. The doses of the two compounds tested are well below the median lethal dose (LD50) for each compound. Furthermore, they are benign, are used as flavorings, perfume components and are present in food (ATSDR, 1995; FDA, 2013; Sigma Aldrich, 2015). Experimental data showed that the release of 2-hexanone and benzaldehyde using the prototype delivery system prevented infestation levels in a dog breed known to be susceptible to parasitism by R. sanguineus s. l. The artificial environmental infestations were established to resemble more closely field conditions of dog exposure to R. sanguineus s. l. (Hansford et al., 2015; Rodríguez-Vivas et al., 2016). Several non-host compounds deter parasitism of animals by hematophagous arthropods (Birkett et al., 2004; Borges et al., 2015; Douglas et al., 2004; Gikonyo et al., 2002, 2003; Pageat, 2005). In several cases, however, their evaluation as the basis for innovative control technologies remains to be accomplished. Saini et al. (2013) tested a collar prototype with repellents identified on the non-host Kobus defassa by Gikonyo et al. (2003) in association with a synthetic repellent against Glossina morsitans. A 90% reduction in the transmission of trypanosomiasis was observed after the use of this prototype.

Refinement of the blend constituents showed the potential to deploy the repellent-based technology to protect cattle from tsetse and the risk of exposure to Trypanosoma spp. (Bett et al., 2015). Pageat (2005) reported the presence of allomones bis (2-ethylhexyll) adipate and 2,2,4-trimethyl-1,3-pentanediol diisobutyrate from the secretion of uropygial gland of ducks that repel red poultry mites, Dermanyssus gallinae, and a product comprising these compounds is being manufactured and distributed in France for use in the control of D. gallinae on poultry farms. The use of a prototype system to deliver repellent compounds provides an opportunity to develop an optimal formulation, in terms of compound quantity and ratio and matrix for formulation stability, for efficient sustained release over several weeks, as has been attempted with other efforts to develop new formulations to control R. sanguineus s. l. infestations on dogs (Bhoopathy et al., 2014; Stanneck et al., 2012). Our results indicated that the counts of larvae (0.47%) and nymphs (12.15%) are a fraction of the total released into the environment where the dogs were housed. Various sources of variability contributed to our inability to recover all the ticks released. Despite our methodical approach, it is possible some ticks were missed during visual and tactile inspection, and during brushing for ectoparasite enumeration, because of the small size of larvae and nymphs, immature ticks can also perish due to desiccation and predation (Apanaskevich and Oliver, 2014; Troughton and Levin, 2007). Host grooming is a preponderant behavior that can result in the removal of up to 80% of the ticks, mainly larvae, because they can attach several times before feeding, which is accompanied by itching that triggers grooming (Hart, 2000; Mooring et al., 1996). Higher counts for adults (180%) were observed parasitizing the dogs than those released in the environment. When the adults were counted, males and unengorged females were not removed from the dogs. Therefore, the higher number of adults found on dogs could be related to the fact that males of R. sanguineus s. l. can survive in the environment for up to 568 days and seek the host repeatedly for feeding, or finding a mate thus moving between hosts and throughout the environment (Hooker et al., 1912; Little et al., 2007; Troughton and Levin, 2007). It is possible that male ticks may have been counted on the same dog in different areas or even on different dogs through time because our prototype formulation was repellent and not acaricidal. Adult tick numbers on treated dogs were lower than on the control animals across all counts. However, this trend was observed for larvae and nymphs only in the fifth count. Synthetic compounds like DEET promote significant repellency against all R. sanguineus s.

l. developmental stages, but the rate and duration of repellency tends to decrease with time when this compound was tested against adults (Kumar et al., 1992). The natural repellents 2-hexanone and benzaldehyde were observed to repel adult R. sanguineus s. l. in vitro as strongly as DEET (Borges et al., 2015). Further research is required to determine the relative repellency of 2-hexanone and benzaldehyde against R. sanguineus s. l. larvae and nymphs. The recovery rate for engorged larvae, nymphs, and females was 0.17, 0.80 and 3.33%, respectively. Notwithstanding the number of engorged tick that may have been missed during brushing, some ticks could have engorged and detached from the dogs before the animals were inspected to quantify the ectoparasite load. Engorged larvae detach from their hosts during the day and night, while engorged nymphs and females detach mainly during the night (Paz et al., 2008). Therefore, the majority of engorged ticks may have dropped off at night before the dogs were brushed during the day. A lack of effect on the biological and reproductive parameters of R. sanguineus s. l. was noted for 2-hexanone and benzaldehyde. However, R. sanguineus s. l. that fed on a resistant dog breed had their biological parameters impaired (Louly et al., 2009). R. sanguineus s. l. apparently senses repellent SLMs to distinguish between resistant and susceptible hosts, but also is deterred by contact compounds produced by resistant animals (Louly et al., 2010). In comparison to chemosensation in congeneric ticks, it was hypothesized that R. microplus could perceive not only phagostimulants known to be present in host blood, but also antifeeding substances that could exist in cattle serum associated with decreased susceptibility to tick infestation (Ferreira et al., 2015). Additional experiments are needed to determine if the deleterious effects observed in R. sanguineus s. l. upon feeding on resistant dogs are caused by compounds other than 2-hexanone and benzaldehyde, which could be present on host skin or circulating in the blood. The specificity of SLM perception and susceptibility to their effects are linked to subtle ratios of the compounds produced by hosts and non-host species (Bruce et al., 2005; Logan et al., 2008; Weldon, 2010). The release rate adopted here roughly matched that observed by Oliveira Filho et al. (2016), where it was shown, using analytical chemistry experiments, that the mean ratio of 2- hexanone: benzaldehyde was ∼1:2 in odor samples collected from beagles. Further work is needed to determine optimal doses and formulations for the two compounds, which will underpin our attempts to develop a commercial product Problems with the control of ticks such as R. sanguineus s. l. and the pathogens they transmit are increasing in complexity (Dantas-Torres, 2015; Esteve-Gassent et al.,

2016). The One Health approach, which is the synthesis of strategies aimed at enhancing animal and human health that take into consideration our environment, has been proposed to address the problem of tick and tick-borne diseases as a way to facilitate the development of sustainable control solutions (Dantas-Torres et al., 2012; Pérez de León et al., 2010; Vayssier-Taussat et al., 2015). Work on natural products that repel arthropod vectors, including ticks, merits attention for development because they could be used as part of integrated pest management programs, and as tools to enable the rational use of acaricides (Guerrero et al., 2014; Mencke, 2013; Pérez de León et al., 2014). Natural semiochemicals such as those tested here are ubiquitous and form part of the adaptive behavioral ecology of R. sanguineus s. l. (Nielsen et al., 2015; Pickett et al., 2010; Weldon, 2010). Further research will help understand the mode of repellency of 2- hexanone and benzaldehyde against R. sanguineus s. l. As has been noted with other species, knowledge gaps on the anatomy and molecular biology of chemosensation and chemoreception in ticks remain to be elucidated (Borges et al., 2016; Esteve-Gassent et al., 2016; Ferreira et al., 2015; Renthal et al., 2016). An enhanced understanding of how ticks detect and process chemical cues to find hosts or avoid unsuitable but potential hosts could offer further innovative technologies to mitigate the burden of these ectoparasites and vectors of pathogens that affect humans, domestic animals, and wildlife.

Acknowledgments

We thank the National Council of Technological and Scientific Development (CNPq), Brazil, for its financial support (Grant no. #/2013-9) and for supply a Scholarship to LMFB (#/2015-0). Rothamsted Research receives grant-aided support from the Biotechnology and Biological Sciences Research Council (BBSRC) of the United Kingdom. USDA is an equal opportunity provider and employer.

References

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CAPÍTULO 4: Persistence and efficacy of a new formulation based on dog allomonal repellents against Rhipicephalus sanguineus sensu lato tick Persistência e eficácia de uma nova formulação baseada em repelentes alomônios de cães contra carrapatos Rhipicephalus sanguineus sensu lato Jaires Gomes de Oliveira Filhoa, Lorena Lopes Ferreiraa, Fernanda de Oliveira Silvaa, Karolina Martins Ferreira Menezesa, Elen Regozino Muniza, Luiza Gabriella Ferreira de Paulaa, André Lucio Franceschini Sarriab, John A. Pickettb, Michael A. Birkettb, Gabriel Moura Mascarinc, Lígia Miranda Ferreira Borgesd a Escola de Veterinária e Zootecnia, Universidade Federal de Goiás. Campus Samambaia, Avenida Esperança, s/n, Campus Universitário, CEP: 74690-900, Goiânia, Goiás, Brazil. E- mails: [email protected]; [email protected];[email protected]; [email protected]; [email protected]; [email protected] b Biointeraction and Crop Protection Department, Rothamsted Research, West Common, Harpenden, Hertfordshire, AL5 2JQ, United Kingdom. E-mails: [email protected];[email protected];[email protected] c.uk c Empresa Brasileira de Pesquisa Agropecuária – Embrapa. Rodovia SP 340, km 127,5, CP 69, CEP: 13820-000, Jaguariúna, São Paulo, Brasil. E-mail: [email protected] d Instituto de Patologia Tropical e Saúde Pública. Universidade Federal de Goiás, Rua 235 s/n, Setor Universitário, CEP: 74605050, Goiânia, Goiás, Brazil. E-mail: [email protected] *corresponding author: [email protected]

Resumo Beagles são menos suscetíveis a carrapatos Rhipicephalus sanguineus sensu lato devido à produção de benzaldeído e 2-hexanona. Nosso trabalho anterior mostrou que esses compostos podem reduzir a carga de carrapatos em cães suscetíveis. Aqui testámos a hipótese de que um aumento na dose destes repelentes e na taxa de liberação poderia aumentar a eficácia e a persistência do efeito repelente. As formulações de liberação lenta destes compostos, com doses mais elevadas e taxas de liberação foram testadas em cães infestados artificialmente. Não se observou aumento significativo na eficácia do repelente com doses mais elevadas e taxas de liberação, enquanto observou-se maior persistência na atividade repelente. O tratamento com as formulações resultou em uma redução de duas a três vezes no número de carrapatos de estágio imaturo por até três semanas. No entanto, o

número de adultos foi semelhante em ambos os grupos. A perda de atividade repelente após a terceira semana de teste coincidiu com uma mudança nas taxas de liberação relativa para os dois compostos. A hipótese é que as quantidades relativas, em vez de quantidades absolutas, de liberação lenta são importantes para a atividade repelente. Então, a hipótese de que a repelência por hospedeiros menos susceptíveis por carrapatos depende da percepção mediada por misturas específicas de voláteis destes hospedeiros. Palavras-chave: Benzaldeído, 2-Hexanona, formulação de liberação lenta, controle de carrapatos

Abstract Beagles are less susceptible to Rhipicephalus sanguineus sensu lato tick due the production of the allomones benzaldehyde and 2-hexanone. Our previous work showed that these compounds can reduce tick burden on susceptible dogs. Here we tested the hypothesis that an increase in repellent dose and release rate could increases repellent efficacy and persistence. Slow-release formulations of these compounds, with higher doses and release rates were tested on artificially infested dogs. No significant increase in repellent efficacy was observed with the higher doses and release rates, whereas a greater persistence in repellent activity was observed. Treatment with the formulations resulted in a two-to-three- fold reduction in the number of immature stage ticks for up to three weeks. However, the number of adults was similar in both groups. Loss of repellent activity after the third week of testing coincided with a marked change in the relative release rates for the two compounds. It is hypothesized that relative amounts, rather than absolute amounts, of repellent release from slow-release formulations are important for repellent activity. Then we hypothesize that the avoidance of less preferred hosts by ticks relies on olfactory- mediated perception of specific blends of volatile cues from less preferred hosts.

Key words: Benzaldehyde, 2-Hexanone, Slow release formulation, Tick control

1. Introduction

The brown dog tick, Rhipicephalus sanguineus sensu lato (Acari: Ixodidae), is an important vector of pathogens for companion animals as well as for human beings (SONENSHINE & ROE, 2014). Management of R. sanguineus currently requires the use of costly acaricides, but the development of resistance in R. Sanguineus s. l. populations has been recently reported (GRAF et al., 2004; EIDEN et al., 2015; RODRIGUEZ-VIVAZ et al., 2017). Therefore, there is an urgent need to find alternative tools for R. Sanguineus s. l. management that either slow the rate of resistance development or remove dependence upon acaricide use altogether. There are several reports that animals of different species or of the same species have different ectoparasitic loads. These differences can be due to variation in the composition of

volatile organic compounds (VOCs) that they emit, thereby signalling to the ectoparasites that animals are more suitable (more susceptible) or less suitable (less susceptible) as hosts (BRUCE et al., 2005; PICKETT et al., 2010). In bovines, Birkett et al. (2004) and Jensen et al. (2004) observed that cattle flies, Haematobia irritans, are differentially attracted to individual cattle within herds, and that differences in VOC emission are responsible for this phenomenon, with less suitable hosts producing higher levels of VOCs (allomones) that confer repellent activity against H. irritans. The production of allomones by the non-host waterbuck, Kobus defassa, against tsetse flies, Glossina morsitans, has also been demonstrated (BETT et al., 2015), and Borges et al. (2015a) demonstrated that larvae of the cattle tick R. microplus are less responsive to the odours of resistant bovine hosts, Bos taurus indicus, than the odours from susceptible Bos taurus taurus. In our seminal work on dog tick / host interactions (LOULY et al., 2009), when comparing the susceptibility of two breeds of dogs to R. sanguineus s. l., it was observed that beagles were less parasitized than English cocker spaniels, and this occurred partially due to the production of allomones by beagles (LOULY et al., 2010; BORGES et al., 2015b). More recently, work elsewhere observed differences in susceptibility to R. sanguineus s. l. on different breeds of dogs, with the English cocker spaniel being more parasitized by this tick when compared to other breeds (SILVA, 2016). Our earlier work on the identification of tick repellents from less-preferred beagles identified two compounds, benzaldehyde and 2-hexanone, in beagle odours, as allomones responsible for the lower susceptibility of these dogs to R. sanguineus s. l. (BORGES et al., 2015b). Using slow-release formulations of the identified allomones, we showed that the mean number of ticks at all stages was significantly lower on treated dog groups than on untreated dog groups (OLIVEIRA FILHO et al. 2017), thus providing the first study of an allomonal repellent formulation for reduction of tick infestation on susceptible dogs. In this new study, we evaluated the persistence and efficacy of a new allomonal formulation, that contained higher doses of benzaldehyde and 2- hexanone, and which therefore emitted higher release rates of the 2 compounds, than those tested by Oliveira Filho et al. (2017), for reducing R. sanguineus s. l. loads on English Cocker Spaniels.

2. Material and methods

2.1. Animals

Ten English cocker spaniels were divided into two groups, treated and untreated, each containing a group of five dogs (four males and one female; 94 ± 10.02 days old). Each group was housed in two stalls (2 m2), with the three smallest animals being housed in one stall and the two largest animals housed in the other. The use of animals (dogs and rabbits) in this study was approved by the Committee on Ethical Animal Use of the Federal University of Goiás (CEUA/UFG, protocol number 024/2014).

2.2. Ticks

Engorged female brown dog ticks, Rhipicephalus sanguineus s. l., were harvested from naturally infested dogs from the municipality of Goiânia, Goiás, Brazil, for the establishment of a tick colony. The ticks were maintained in an acclimatized chamber (27 ± 1 ◦C and 80% R.H.) and fed on rabbits, Oryctolagus cuniculus, to obtain differing life stages for use in dog infestations. The ticks used in the experiments were aged between one and three weeks old. Rabbits were removed from use after two consecutive infestations. During infestations, rabbits were examined daily and none showed symptoms of damage due to tick parasitism.

2.3. Slow-release formulations

Sheets of cellulose sponge were treated with benzaldehyde (2 ml) and 2-hexanone (1 ml) and heat-sealed in polyethylene tubing (1000 gauge). The mean release rate per day of the compounds from the sachet formulations was determined over five weeks using dynamic headspace collection (air entrainment) and GC analysis. Thus, three sachets filled with either benzaldehyde or 2-hexanone were enclosed in a glass vessel (700 mL). Air was pumped through an activated charcoal filter into the vessel (1 L/min) and was then drawn (500 cc/min) into tubes containing the adsorbent Porapak Q (50 mg). After one hour, volatiles collected on the Porapak Q were eluted with 750 uL of redistilled diethyl ether and the samples were stored at −20 ◦C until required for GC analysis. Extracts (1 uL) were analysed

on an Agilent 6890 N GC fitted with a 10 m × 0.32 mm i.d. HP-1 column. The oven temperature was maintained at 30 ◦C for 0.1 min, then programmed at 10 ◦C min−1 to 250 ◦C and held for 30 min. It was observed that for benzaldehyde, the release was more or less constant and varied between 48.25 to 61.82 μg/day. However, for 2-hexanone, the release was only constant in the first 3 weeks, ranging from 38.33 to 55.28 μg/day, decreasing sharply in the fourth week to 8.53 μg/day. The release ratio between benzaldehyde and 2- hexanone ranged from 1.18 to 1.61 in the first 3 weeks, which was reasonably close to that found naturally in beagles by Oliveira Filho et al. (2016). In the fourth week, the ratio was 6.41 and different from that observed in Oliveira Filho et al. (2016).

2.4. Tick infestation and counting

Every week, for 35 days, 4000 larvae, 200 nymphs and 60 adults (30 males and 30 females) were released onto the walls of each stall, totalling six artificial infestations. Collars containing the sachets with benzaldehyde and 2-hexanone were placed simultaneously with the second infestation and withdrawn after the fifth infestation. Two days after the first infestation, dogs were inspected once a day at 16:00h for 40 days. Dogs were simultaneously inspected by two trained personnel for tick counts using a magnifying glass, 1.5x to 10x, with a led light (Led headhand magnifier model MG811007- A, China) to help visualize the ticks. Ticks were not removed during inspections. This experiment was carried out in September and October 2016, where the temperature measured was between 31ºC and 19ºC.

2.5. Statistical analysis

Count data for larvae, nymphs and adults were separately fitted to a negative binomial model, since this model gave the best goodness-of-fit based on AIC and half- normal plot criteria for model selection. Treatment, infestation date and their interaction term were included in the model as predictors. Significant differences between treated vs. control dogs were determined by Likelihood Ratio (LR) chi-square test for each infestation date. Analysis was carried out using R statistical software environment (R Core Team, 2015; http://www.R-project.org/).

3. Results

In the week before animals received repellent collars, the number of larvae, nymphs and adults was similar in treated and control groups (larvae p=0,3392; nymphs p=0.8853; adults p=0.5137). The same was observed after the sixth infestation, when collars had been removed (larvae p=0.6765; nymphs p=0.5019; adults p=0.5335) (Fig. 1). For the weeks during the experiment when dogs wore collars, a significant effect on the number of nymphs and larvae was observed. During the first two weeks after collar attachment, which corresponded to the 2nd and 3rd infestations respectively, the number of larvae in the treated group was 2-3 fold lower than in the control group (2nd infestation p=0.01; 3rd infestation p=0.0003). In the third week after attachment, although the number of larvae was lower in the treated group than in the control, there was no statistical difference (p=0.054). The number of nymphs in the treated group was almost half of the number in the control group during the three weeks after collar attachment (2nd infestation p=0.0001; 3rd infestation p=0.008; 4th infestation p<0.0001). However, the number of adults was similar in both groups across all infestations (Fig.1).

Fig. 1. Mean (±SE, standard error) number of ticks (larvae, nymphs, and adults) retrieved from untreated (control) and treated (repellent collar) dogs across infestation periods. The repellent collars were attached on the 2nd week and dogs used them for 4 weeks (indicated by vertical arrows). Individual data points were shown per week. Significant difference between control and treated dogs is indicated by P < 0.05 (*) or P < 0.01 (**).

Overall, tick loads on dogs wearing the collars containing the repellent formulation were significantly lower for larvae (p=0.0123) and nymphs (p=0.000267) when compared to untreated dogs from the control group. However, for adults, the numbers found in both groups was virtually the same i.e. 808 in the treated group and 807 in the control group (P=0.9612) (Fig. 2).

Fig. 2. Overall mean (±SE, standard error) number of ticks (larvae, nymphs, and adults) retrieved from untreated (control) and treated (repellent collar) dogs. Individual data points are shown. Significant difference between control and treated dogs is indicated by P < 0.05 (*) or P < 0.01 (**)

4. Discussion

The results in this study corroborate results from our earlier work (OLIVEIRA FILHO et al. 2017), where it was shown that slow-release formulations of benzaldehyde and 2-hexanone, produced by beagles that are less preferred by brown dog ticks, R. sanguineus s. l., can reduce tick loads on susceptible dogs. However, differences between this study and the earlier work were observed. In our earlier work, we observed repellency against adults across all weeks, although in lower rates than observed for the immature stages. In this study, however, no repellency of adults was observed across any of the weeks. The number of adults released on the walls, and higher counts than the actual number released, was observed in both experiments. It is possible that the same male tick may have been counted either on the same dog in different areas or on different dogs through time, or that nymphs have engorged on dogs and have moulted during the experiment, thereby increasing the number of adults attached on the dogs. Future work needs to involve more reliable evaluation, including removal of ticks from hosts after counting, as suggested elsewhere (MARCHIONDO et al. 2013). Furthermore, although similar repellency of the immature stages of R. sanguineus s. l. was observed, a higher persistence of the repellent effect was observed in this study. Formulations remained active for the first three weeks, whereas they were only active for one week in the previous work. The difference in persistence between the two studies may be because in the current study, a higher dose and release rate of the repellent compounds was used. However, what cannot be completely ruled out is that the observed greater persistent effect could be due to an increase in the number of ticks released in the experiments and an improvement in the counting of immature tick stages, compared to Oliveira Filho et al. (2017). Further work is required to rule out the effect of tick numbers. During the first three weeks of the study, the ratio of benzaldehyde: 2-hexanone release from the formulations was roughly similar to that found naturally in beagles (OLIVEIRA FILHO et al. 2016). However, from the 4th week, the ratio of release was markedly different, which coincided with a reduction in tick repellency. In the study, the repellents were released in a background of attractant VOCs from English Cocker Spaniel dogs, with the dogs being perceived, from an olfactory perspective, as less-preferred beagles (INOKUMA et al., 1997; JITAPALAPONG et al., 2000; LOULY et al., 2009; LOULY et al., 2010). From the results, we hypothesize that relative amounts, rather than absolute

amounts, of repellent release from the slow-release formulations, are important for tick repellent activity, and that avoidance of less-preferred hosts by ticks relies on olfactory- mediated perception of specific blends of volatile cues from less preferred hosts. Although beagles are considered as less preferred by R. sanguineus s. l., they are still naturally parasitized (LORUSSO et al., 2010; DANTAS-TORRES & LORUSSO, 2011). Based on the results in our study, we propose that repellent allomones produced by beagles can decrease loads of R. sanguineus s. l. on susceptible dogs, but that they cannot control ticks satisfactorily by themselves. The push-pull strategy, also known as the stimulo- deterrent diversionary strategy, has been described as a route by which to manage animal pests, and consists of two distinct phases: a push phase which aims at repelling or deterring parasites of resources such as animals, through the use of stimuli that act as repellents for these parasites or mask the presence of the host, and a pull phase, used simultaneously, which aims to use either highly attractive stimuli that may indicate host presence, or pheromones, to attract and divert the parasites of the resources to be protected. Currently, this strategy is being developed for the sustainable management of Savannah spp. tsetse flies that vector the trypanosomiasis parasite affecting millions of cows in sub-Saharan Africa (COOK et al., 2007; www.icipe.org/impacts/). To our knowledge, this type of strategy has yet to be attempted for the management of ectoparasitic ticks affecting companion animals. New experiments are needed to explore the possibility of using repellent formulations in conjunction with an attractant trap and an acaricide, as proposed in the push-pull strategy, for R. sanguineus s. l. management on domestic dogs.

5. Conclusion The relative amount, rather than absolute amounts, of dog allomones, benzaldehyde and 2- hexanone release from slow-release formulations, are important for repellent activity and can decrease the load of R. sanguineus s. l. immatures on susceptible dogs.

Acknowledgments We thank the National Council of Technological and Scientific Development (CNPq), Brazil, for its financial support (Grant no. #/2013-9) and for supply a Scholarship to LMFB (#/2015- 0). Rothamsted Research receives grant-aided support from the Biotechnology and Biological Sciences Research Council (BBSRC) of the United Kingdom.

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CAPÍTULO 5: Considerações Finais

R. sanguinueus s. l. é sem duvida uma das espécies de grande relevância tanto para a saúde publica quanto para a saúde animal, portanto encontrar alternativas para seu controle são necessárias devido principalmente ao grande problema de resistência desta espécie aos acaricidas químicos. Nos trabalhos aqui apresentados demonstrou-se a avaliação e quantificação de 2-hexanona e benzaldeido, em quantidades relativamente significativas nos odores de beagles, durante um período de uma semana. Sendo ainda capaz de demonstrar que tais compostos estão persistentemente presentes nos odores destes cães o que os torna um hospedeiro resistente para o carrapato R. Sanguineus s. l.. Também foi observado que benzaldeído aparece frequentemente em uma concentração maior que 2-hexanona, sendo ainda que esta razão média variou 1,1:1 pra 3,6:1 com uma média final de 2,2:1, benzaldeído pra 2-hexanona respectivamente. Sendo ainda observado no trabalho posterior que o efeito dos compostos emitidos por hospedeiros resistentes podem ser transferidos para hospedeiros susceptíveis por meio de elaboração de formulações com estes compostos. Onde verificou-se pela primeira vez a diminuição de uma infestação de R. Sanguineus s. l. mediante o uso de uma mistura de benzaldeído 2-hexanona (na razão de 2:1) sobre hospedeiros susceptíveis. Contudo ao se aumentar as concentrações e as razões destas formulações de liberação lenta. Não observou-se maior eficácia mas sim uma maior persistência na atividade repelente. Sendo ainda que a perda de atividade repelente de formulações de liberação lenta após a terceira semana de teste coincidiu com uma mudança nas taxas de liberação relativa para os dois compostos. Os resultados aqui apresentados demonstram a atividade in vivo de uma formulação contendo repelentes de um hospedeiro resistente, o que pode ser considerado como uma ferramenta mais segura mais eficaz devido ao seu poder ecológico, para ser usada para em infestações de R. sanguineus s. l.. Sendo que ficou claro nos trabalhos aqui realizado a importância da razão entre os compostos ou seja do valor relativo entre estes e não apenas o valor absoluto. Sendo ainda recomendado associação de outras formas de controle, haja visto que mesmo cães beagles que são hospedeiros resistentes são encontrados naturalmente infestados.

ANEXO I

Anexo II

Anexo III