Identification of anti-horn fly vaccine antigen candidates using a reverse vaccinology approach.

BACKGROUND: The horn fly, Haematobia irritans irritans, causes significant production losses to the cattle industry. Horn fly control relies on insecticides; however, alternative control methods such as vaccines are needed due to the fly's capacity to quickly develop resistance to insecticides, and the pressure for eco-friendly options. METHODS: We used a reverse vaccinology approach comprising three vaccine prediction and 11 annotation tools to evaluate and rank 79,542 translated open reading frames (ORFs) from the horn fly's transcriptome, and selected 10 transcript ORFs as vaccine candidates for expression in Pichia pastoris. The expression of the 10 selected transcripts and the proteins that they encoded were investigated in adult flies by reverse transcription polymerase chain reaction (RT-PCR) and mass spectrometry, respectively. Then, we evaluated the immunogenicity of a vaccine candidate in an immunization trial and the antigen's effects on horn fly mortality and fecundity in an in vitro feeding assay. RESULTS: Six of the ten vaccine candidate antigens were successfully expressed in P. pastoris. RT-PCR confirmed the expression of all six ORFs in adult fly RNA. One of the vaccine candidate antigens, BI-HS009, was expressed in sufficient quantity for immunogenicity and efficacy trials. The IgG titers of animals vaccinated with BI-HS009 plus adjuvant were significantly higher than those of animals vaccinated with buffer plus adjuvant only from days 42 to 112, with a peak on day 56. Progeny of horn flies feeding upon blood from animals vaccinated with BI-HS009 plus adjuvant collected on day 56 had 63% lower pupariation rate and 57% lower adult emergence than the control group (ANOVA: F (1, 6) = 8.221, P = 0.028 and F (1, 6) = 8.299, P = 0.028, respectively). CONCLUSIONS: The reverse vaccinology approach streamlined the discovery process by prioritizing possible vaccine antigen candidates. Through a thoughtful process of selection and in vivo and in vitro evaluations, we were able to identify a promising antigen for an anti-horn fly vaccine.

Susceptibility of field populations of Haematobia irritans to fipronil in Uruguay / Suscetibilidade de populações de campo de Haematobia irritans ao fipronil no Uruguai

Fipronil was registered in Uruguay in 1997, and, since then, it has been used for the control of Haematobia irritans irritans and Rhipicephalus microplus. The susceptibility of H. irritants to this drug has not been evaluated. Therefore, the goal of the present study was to evaluate the resistance of H. irritans to fipronil. Additionally, a survey was carried out with the farmers to evaluate the use of fipronil for H. irritans control in the ranches where the flies came from. For the bioassays, 31 field populations of H. irritans were exposed to 10 concentrations of fipronil (3.2-16.0μg.cm2), and their LC50 values were calculated using probit analysis. A bioassay was performed with horn flies from the susceptible colony maintained at the USDA-ARS Knipling-Bushland U.S. Livestock Insects Research Laboratory for comparison and calculation of resistance ratios (RRs). All 31 field populations surveyed in the study were susceptible to fipronil, with resistance ratios ranging from <0.5 to 2.2. Four populations with RRs >1 did not differ significantly from the susceptible strain. A single population showed an RR >2.2. Overall, the survey shows that fipronil was mostly used for R. microplus control, and in only three ranches, which were free of R. microplus, was fipronil used for horn fly control. Seventeen farmers did not use fipronil at all in the last three years. It is concluded that, in Uruguay, field populations of horn flies remain susceptible to fipronil.(AU)

O fipronil foi registrado no Uruguai em 1997 e, desde então, tem sido utilizado no controle de Haematobia irritans irritans e Rhipicephalus microplus. O objetivo do presente estudo foi avaliar a susceptibilidade de populações de campo de H. irritans ao fipronil. Além disso, foi realizada uma pesquisa para avaliar a utilização de fipronil e as práticas de controle de H. irritans nas fazendas de onde provinham as moscas. Para os bioensaios, 31 populações de campo de H. irritans foram expostas a 10 concentrações de fipronil (3,2-16,0μg.cm2), e seus valores de CL50 foram calculados usando análise probit. Um bioensaio foi realizado com H. irritans da colônia suscetível mantida no USDA-ARS Knipling-Bushland U.S. Livestock Insects Research Laboratory para comparação e cálculo das razões de resistência (RRs). Todas as 31 populações de campo pesquisadas no estudo eram suscetíveis ao fipronil, com taxas de resistência variando de <0,5 à 2,2. Quatro populações com Rrs >1 não diferiram significativamente da cepa suscetível. Uma única população apresentou RR >2,2. No geral, o fipronil tinha sido usado principalmente para o controle de R. microplus, e em apenas três fazendas, que estavam livres de R. microplus, o fipronil era utilizado para o controle da H. irritans. Em 17 fazendas não tinha sido utilizado fipronil nos últimos três anos. Conclui-se que no Uruguai as populações de H. irritans no campo permanecem suscetíveis ao fipronil.(AU)

Susceptibility of field populations of Haematobia irritans to fipronil in Uruguay

ABSTRACT: Fipronil was registered in Uruguay in 1997, and, since then, it has been used for the control of Haematobia irritans irritans and Rhipicephalus microplus. The susceptibility of H. irritants to this drug has not been evaluated. Therefore, the goal of the present study was to evaluate the resistance of H. irritans to fipronil. Additionally, a survey was carried out with the farmers to evaluate the use of fipronil for H. irritans control in the ranches where the flies came from. For the bioassays, 31 field populations of H. irritans were exposed to 10 concentrations of fipronil (3.2-16.0g.cm2), and their LC50 values were calculated using probit analysis. A bioassay was performed with horn flies from the susceptible colony maintained at the USDA-ARS Knipling-Bushland U.S. Livestock Insects Research Laboratory for comparison and calculation of resistance ratios (RRs). All 31 field populations surveyed in the study were susceptible to fipronil, with resistance ratios ranging from 0.5 to 2.2. Four populations with RRs >1 did not differ significantly from the susceptible strain. A single population showed an RR >2.2. Overall, the survey shows that fipronil was mostly used for R. microplus control, and in only three ranches, which were free of R. microplus, was fipronil used for horn fly control. Seventeen farmers did not use fipronil at all in the last three years. It is concluded that, in Uruguay, field populations of horn flies remain susceptible to fipronil.

RESUMO: O fipronil foi registrado no Uruguai em 1997 e, desde então, tem sido utilizado no controle de Haematobia irritans irritans e Rhipicephalus microplus. O objetivo do presente estudo foi avaliar a susceptibilidade de populações de campo de H. irritans ao fipronil. Além disso, foi realizada uma pesquisa para avaliar a utilização de fipronil e as práticas de controle de H. irritans nas fazendas de onde provinham as moscas. Para os bioensaios, 31 populações de campo de H. irritans foram expostas a 10 concentrações de fipronil (3,2-16,0g.cm2), e seus valores de CL50 foram calculados usando análise probit. Um bioensaio foi realizado com H. irritans da colônia suscetível mantida no USDA-ARS Knipling-Bushland U.S. Livestock Insects Research Laboratory para comparação e cálculo das razões de resistência (RRs). Todas as 31 populações de campo pesquisadas no estudo eram suscetíveis ao fipronil, com taxas de resistência variando de 0,5 à 2,2. Quatro populações com Rrs >1 não diferiram significativamente da cepa suscetível. Uma única população apresentou RR >2,2. No geral, o fipronil tinha sido usado principalmente para o controle de R. microplus, e em apenas três fazendas, que estavam livres de R. microplus, o fipronil era utilizado para o controle da H. irritans. Em 17 fazendas não tinha sido utilizado fipronil nos últimos três anos. Conclui-se que no Uruguai as populações de H. irritans no campo permanecem suscetíveis ao fipronil.

Impacts of long-term insecticide treatment regimes on skdr and kdr pyrethroid resistance alleles in horn fly field populations.

We evaluated the effects of four different 6-year duration control strategies on the resistance levels and frequency of the pyrethroid target site resistance alleles, superkdr (skdr) and kdr, at four field populations of Haematobia irritans irritans (Linnaeus, 1758) (Diptera: Muscidae) in Louisiana, USA. Consecutive use of pyrethroid ear tags for 6 years caused a significant increase in the resistance ratio to pyrethroids as well as the frequencies of both skdr and kdr resistance alleles. After 3 years of consecutive use of pyrethroid ear tags, followed by 1 year with no treatment, and followed by 2 years with organophosphate ear tags, the resistance ratio for pyrethroid was not significantly affected, the %R-skdr significantly dropped while the %R-kdr allele remained relatively high and stable. Similar results were observed when pyrethroid ear tags were used for three consecutive years, followed by 1 year with no treatment, and followed by 2 years with endosulfan ear tags; however, this treatment resulted in a slight increase in the resistance ratio for pyrethroids. In a mosaic, the resistance ratio for pyrethroids showed a 2.5-fold increase but the skdr-kdr genetic profiles did not change, as the %R alleles (skdr and kdr) remained low and stable through the 6 years. Lack of exposure to pyrethroid insecticides for 3 years significantly affected the skdr mutation but not the kdr mutation, preventing re-establishment of susceptibility to pyrethroids. SS-SR (skdr-kdr) individuals were responsible for the maintenance of the kdr mutation in two of the populations studied, and fitness cost seems to strongly affect the SR-RR genotype. None of the four treatment regimens evaluated in the study had satisfactory results for the management of kdr resistance alleles.

The assembled transcriptome of the adult horn fly, Haematobia irritans.

The horn fly, Haematobia irritans irritans (Linnaeus, 1758; Diptera: Muscidae), a hematophagous external parasite of cattle, causes considerable economic losses to the livestock industry worldwide. This pest is mainly controlled with insecticides; however, horn fly populations from several countries have developed resistance to many of the products available for their control. In an attempt to better understand the adult horn fly and the development of resistance in natural populations, we used an Illumina paired-end read HiSeq and GAII approach to determine the transcriptomes of untreated control adult females, untreated control adult males, permethrin-treated surviving adult males and permethrin + piperonyl butoxide-treated killed adult males from a Louisiana population of horn flies with a moderate level of pyrethroid resistance. A total of 128,769,829, 127,276,458, 67,653,920, and 64,270,124 quality-filtered Illumina reads were obtained for untreated control adult females, untreated control adult males, permethrin-treated surviving adult males and permethrin + piperonyl butoxide-treated killed adult males, respectively. The de novo assemblies using CLC Genomics Workbench 8.0.1 yielded 15,699, 11,961, 2672, 7278 contigs (≥ 200 nt) for untreated control adult females, untreated control adult males, permethrin-treated surviving adult males and permethrin + piperonyl butoxide-treated killed adult males, respectively. More than 56% of the assembled contigs of each data set had significant hits in the BlastX (UniProtKB/Swiss-Prot database) (E <0.001). The number of contigs in each data set with InterProScan, GO mapping, Enzyme codes and KEGG pathway annotations were: Untreated Control Adult Females - 10,331, 8770, 2963, 2183; Untreated control adult males - 8392, 7056, 2449, 1765; Permethrin-treated surviving adult males - 1992, 1609, 641, 495; Permethrin + PBO-treated killed adult males - 5561, 4463, 1628, 1211.

Simultaneous detection of pyrethroid, organophosphate, and cyclodiene target site resistance in Haematobia irritans (Diptera: Muscidae) by multiplex polymerase chain reaction.

The horn fly, Haematobia irritans irritans (L., 1758) (Diptera: Muscidae), is an important pest that causes significant economic losses to the livestock industry, but insecticide resistance in horn fly populations has made horn fly control increasingly difficult to achieve. In this study, we developed a multiplex polymerase chain reaction (PCR) assay to simultaneously detect target site resistance to pyrethroids (kdr mutation), organophosphates (G262A acetylcholinesterase mutation), and cyclodienes (Rdl mutation) and used the new procedure to follow the progression of these three mutations after exposure to different insecticide pressure. We assayed flies collected at the Macon Ridge research station, Winnsboro, LA, from 2008 to 2012. The multiplex PCR showed robust results in all our assays. The kdr mutation remained at high frequencies during all years, even after 4 yr with no use of pyrethroids. The G262A acetylcholinesterase mutation fluctuated from 7.5 to 23.8% during the studied years, while the Rdl mutation was rare in 2008, 2009, and June 2010, and then significantly increased after the first use of endosulfan. The possibility of screening for all the known target site resistance mutations in a single PCR reaction makes the multiplex PCR a useful and affordable tool that can be used to help diagnose insecticide resistance.

Discovery of the Rdl mutation in association with a cyclodiene resistant population of horn flies, Haematobia irritans (Diptera: Muscidae).

The horn fly, Haematobia irritans irritans, is an obligate blood-feeding parasite of cattle that causes significant economic impact in many countries. We investigated the resistance of a horn fly population from Louisiana/USA to endosulfan, a cyclodiene insecticide. Bioassays were performed in 2010 and 2011 in order to determine the resistance ratio of the population to endosulfan and a PCR assay was developed to detect the Rdl mutation which is the replacement of an alanine with a serine at the GABA receptor locus that has been associated with resistance to cyclodienes in other insect species. Endosulfan tags had provided 8 weeks of effective control in 2010 but only 1 week in 2011. After only one summer (June-September/2010) of exposure to the endosulfan tagged cattle, there was a significant increase in the resistance ratio for endosulfan in the fly population. Most flies surveyed by the PCR diagnostic assay were homozygous susceptible at the Rdl locus, the resistant (R) allele was mainly present in the heterozygous state and there was no difference in the frequency of the R allele between female and male flies. After the first year's exposure of the horn flies to the endosulfan tags, the frequency of the R allele increased significantly. However, after one year without endosulfan treatment (2011-2012), the frequency of the R allele significantly dropped. These results indicate that target site resistance was responsible, at least in part, for the resistance and that a fitness cost is possibly associated with the Rdl mutation.

Clinical aspects and dynamics of auricular parasitosis in Gir cattle / Aspectos clínicos e dinâmica de parasitoses auriculares em bovinos da raça Gir

We evaluated the dynamics of ear infestations caused by Rhabditis spp. and Raillietia spp., which were correlated with animal age, intensity of clinical signs and climate factors. Sixty-four Gir cattle were distributed into three groups: GA - 23 calves with 4 to 6 months of age; GB - 18 calves with 7 to 12 months of age; and GC - 23 heifers with 13 to 33 months of age. Five samplings, defined as S1, S2, S3, S4 and S5 were performed every three months from August 2008 to August 2009. The ear secretion was collected using the auricular washing method for the right ear and a swab for the left ear. A clinical assessment of the animals was performed, and they were classified according to the presence and severity of otitis. The highest relative frequency of rhabditosis was 52.2% in GC at the last sampling. In the first sampling, 42.2% of the animals were infested by Raillietia spp. The older cattle were more susceptible to infestations by both parasites. No correlation of Rhabditis spp. and Raillietia spp. parasitism with climate factors was found. The results showed that both parasites could infest Gir cattle, and in most cases, there was no co-infestation. Only older animals parasitized by the nematode showed clinical signs of the disease.

Avaliou-se a dinâmica da infestação auricular causada por Rhabditis spp. e por Raillietia spp., relacionando-a com a idade dos animais, com a intensidade dos sinais clínicos e com os fatores climáticos. Utilizaram-se 64 bovinos Gir, distribuídos em três grupos: GA - 23 bezerros de quatro a seis meses de idade, GB - 18 bezerros de sete a 12 meses e GC - 23 novilhas de 13 a 33 meses. Foram feitas cinco coletas a cada três meses. A secreção auricular foi coletada utilizando o método de lavagem auricular na orelha direita e um swab na orelha esquerda. Fez-se avaliação clínica dos animais classificando-os de acordo com a presença e gravidade da otite. A maior frequência relativa da rhabditiose encontrada foi de 52,2%, no GC na ultima coleta. Na primeira coleta, 42,2% dos animais estavam infestados pela Raillietia spp. Os bovinos mais velhos foram mais suscetíveis às infestações de ambos os parasitos. Não foi encontrada correlação entre o parasitismo por Rhabditis spp. e por Raillietia spp. e os fatores climáticos. Concluiu-se que ambos os parasitas podem infestar bovinos da raça Gir e na maioria dos casos, não ocorreram infestações simultâneas. Apenas os animais mais velhos, parasitados pelo nematóide, apresentaram sinais clínicos da doença.

Caracterização do controle de Haematobia irritans e Rhipicephalus (Boophilus) microplus no Triângulo Mineiro e Alto Paranaíba, Minas Gerais / Characterization of Haematobia irritans and Rhipicephalus (Boophilus) microplus control in Triângulo Mineiro and Alto Paranaíba, Minas Gerais

O controle eficaz de Haematobia irritans (mosca-dos-chifres) e Rhipicephalus (Boophilus) microplus (carrapato-do-boi) é um fator crucial à maior rentabilidade da pecuária brasileira, porém ainda constitui-se um desafio. Um maior conhecimento das práticas adotadas no combate destes parasitos faz-se necessário para que se possam estruturar estratégias de controle mais próximas da realidade do produtor rural e mais fáceis de serem executadas. Este estudo caracterizou, através de entrevistas, as práticas adotadas no controle desses ectoparasitos em 23 propriedades da mesorregião do Triângulo Mineiro e Alto Paranaíba, Minas Gerais. A maioria dos entrevistados reconheceu a importância desses parasitos para a atividade pecuária, entretanto, diversos problemas relativos ao controle parasitário foram observados. O controle da mosca-dos-chifres e do carrapato-do-boi era uma prática rotineira em, respectivamente, 17,4% e 95,7% das propriedades, geralmente realizado em função de elevado grau de infestação dos animais. Mais de seis aplicações de ectoparasiticidas eram realizadas por ano em todas as propriedades que empregavam tratamentos para o controle da mosca e em 76,5% daquelas que combatiam o carrapato. Os produtos eram aplicados principalmente com bombas costais manuais (63,6%) e sem a contenção dos animais em 45,5% das propriedades. A diluição dos produtos segundo recomendações dos fabricantes era realizada por 45,4% dos entrevistados, porém, 63,6% aplicavam um volume de solução por animal menor do que o tecnicamente recomendado. Utilizava-se principalmente a associação de piretróides e organofosforados para o controle de ambos os parasitos. Apesar de utilizar rotineiramente ectoparasiticidas, o uso de equipamento de proteção individual (EPI) não era comum entre os entrevistados. A maioria dos entrevistados conhecia algumas características epidemiológicas dos parasitos, entretanto, o controle parasitário adotado na maior parte das propriedades tende a comprometer não apenas a eficácia dos tratamentos, mas a suscetibilidade dos parasitos e a sustentabilidade do controle.

The effective control of Haematobia irritans (horn fly) and Rhipicephalus (Boophilus) microplus (cattle tick) is crucial for increasing the productivity of livestock industry in Brazil, but still remains a challenge. A greater knowledge about parasite control in the field is necessary to establish control strategies closer to farmer's reality and easier to be performed. This study characterized the practices adopted for controlling such ectoparasites on 23 cattle ranches from the Triângulo Mineiro and Alto Paranaíba region in the state of Minas Gerais, through interviews. Most interviewees recognized the importance of these parasites to livestock, however, many problems related to parasite control were observed. Control of horn flies and cattle tick was a routine in 17.4% and 95.7% of the ranches, respectively, triggered by high infestations on animals. More than six insecticide/acaricide treatments were applied yearly in all cattle ranches controlling horn flies and in 76.5% of the ranches controlling the cattle tick. Pesticide products were applied mostly by manual backpack sprayers (63.6%) without restraining the animals in 45.5% of ranches. Product dilution following manufacturer's recommendations was performed by 45.5% of interviewees, but 63.6% of them used lower doses per animal than technically recommended. Associations of organophosphates and pyrethroids were the main products used to control both parasites. Although pesticide use was a routine practice, the use of personal protective equipment (PPE) was not common among surveyed people. Most interviewees were able to recognize epidemiological characteristics of the parasites; however, parasite control measures adopted at most cattle ranches tend to jeopardize the efficacy of products, susceptibility of parasites and sustainability of control programs.

Coxiella symbiont in the tick Ornithodoros rostratus (Acari: Argasidae).

In the present study, the presence of tick-associated bacteria and protozoa in Ornithodoros rostratus ticks (adults, nymphs, and eggs) from the Pantanal region of Brazil were determined by molecular detection. In these ticks, DNA from protozoa in the genera Babesia and Hepatozoon, and bacteria from the genera Rickettsia, Borrelia, Anaplasma, and Ehrlichia were not detected. Conversely, all tested ticks (100%) yielded PCR products for 3 Coxiella genes (16S rRNA, pyrG, cap). PCR and phylogenetic analysis of 3 amplified genes (16S rRNA, pyrG, cap) demonstrated that the agent infecting O. rostratus ticks was a member of the genus Coxiella. This organism grouped with Coxiella symbionts of other soft tick species (Argasidae), having different isolates of C. burnetii as a sister group, and these 2 groups formed a clade that grouped with another clade containing Coxiella symbionts of hard tick species (Ixodidae). Analysis of tick mitochondrial 16S rRNA gene database composed mostly of tick species previously shown to harbor Coxiella symbionts suggests a phylogenetic congruence of ticks and their Coxiella symbionts. Furthermore, these results suggest a very long period of coevolution between ticks and Coxiella symbionts and indicates that the original infection may have occurred in an ancestor common to the 2 main tick families, Argasidae (soft ticks) and Ixodidae (hard ticks). However, this evolutionary relationship must be confirmed by more extensive testing of additional tick species and expanded populations.

A novel Rickettsia infecting Amblyomma dubitatum ticks in Brazil.

A total of 130 adult free-living ticks of Amblyomma cajennense and 9 free-living Amblyomma dubitatum were collected in the surroundings of the Pampulha Lake, within Belo Horizonte city, state of Minas Gerais, south-eastern Brazil. Each adult tick was tested for rickettsial infection by PCR protocols targeting the rickettsial genes gltA, htrA, and ompA. All the 130 A. cajennense ticks were negative by PCR. In contrast, all 9 A. dubitatum ticks were shown to contain rickettsial DNA. PCR products were sequenced, generating identical sequences for each gene among the 9 ticks, which were shown to contain a novel agent, here designated as Rickettsia sp. strain Pampulha. Phylogenetic analyses inferred from the 3 rickettsial genes (gltA, htrA, ompA) showed that Rickettsia sp. strain Pampulha segregated in the same cluster with R. tamurae, R. monacensis, and several other unclassified rickettsial strains; however, strain Pampulha is a unique Rickettsia strain for the New World, since its closest relatives, according to 3 genes (gltA, htrA, and ompA), are all from the Old World. Because A. dubitatum eventually bites humans, further studies are necessary to determine the potential pathogenicity of Rickettsia sp. strain Pampulha to humans.

Biological parameters of ticks (Amblyomma cajennense Fabricius, 1787) under field and laboratory conditions in Pedro Leopoldo, State of Minas Gerais, Brazil.

This study evaluated the biological parameters of the duration of the pre-oviposition and egg incubation periods and maximum survival of unfed larvae, under field and laboratory conditions, with the aim of providing support for more rational measures for controlling Amblyomma cajennense. This study was conducted at the Experimental Farm of the Veterinary School of the Federal University of Minas Gerais. Every 14 days, from September 1997 to March 1998 and from October 1998 to April 1999, six netted tubes containing engorged females were kept on pasture of Brachiaria decumbens that was up to 60 cm high and another six were kept in a BOD glass chamber (27 degrees C, 80% relative humidity). For both groups, 84 and 72 females were used during the first and second observation periods, respectively. The duration of the pre-oviposition period was similar under both conditions (5-7 days). Significant differences were seen between the duration of the egg incubation period under field conditions (56 to 60 days) and under BOD conditions (34 to 39 days); and between the maximum survival of the unfed larvae under field conditions (14 months) and under BOD conditions (9.5 months). These suggest that behavioral diapause was occurring. The long survival of unfed larvae in pasture needs to be considered when implementing Amblyomma cajennense control programs.

[Control of Anocentor nitens (Neumann, 1897) (Acari: Ixodidae) on equines]. / Controle de Anocentor nitens (Neumann, 1897) (Acari: Ixodidae) em equinos.

This trial evaluated control practices of Anocentor nitens on equines, using spraying devices and application of acaricide paste formulation in the auricular pavilion and nasal diverticulum. The study was carried out from October 2003 to March of 2008 and the evaluations had been divided in the following stages: Phase 1--out/03 mar/04 and Phases 2, 3, 4 and 5, respectively, correspondents to the month's periods until março/08. It was used score of 0 to 3 to classify infestation levels. From abr/04 to mar/06 was implanted a schedule of acaricide sprayings every seven days and divided in two series. The first one beginning in April 2004 and the second beginning in July, both using six sprayings treatments with pyrethroid chemical base--cypermethrin 0,015%, plus topical treatments applied monthly in the auricular pavilions (powder acaricide). From abril/06 to março/08 was carried out similar schedule treatments, each two months, using an experimental acaricide paste in the auricular pavilion and nasal diverticulum. Phases 2 and 3 did not showed reduction of the parasitic loads of A. nitens compared to the control period. Whereas in Phases 4 and 5 registered significant reduction compared control period and also with the results of Phases 2 and 3, characterizing the effectiveness of the treatment with the acaricide paste formulation. Results demonstrated of A. nitens populations present in the nasal diverticulum are important in the maintenance of the infestations on equines, and necessary attention to this anatomical structure when controlling ticks.

[Effect of the urea fertilization in pasture on Rhipicephalus (Boophilus) microplus (Acari: Ixodidae)]. / Efeito da adubação com uréia em pastagem, sobre Rhipicephalus(Boophilus) microplus (Acari: Ixodidae).

The objective of this study was to verify the effect of the urea fertilization in pasture on Rhipicephalus (Boophilus) microplus population in free living stage. Two groups had been made, a control group and a urea treatment group, each group with 10 lots of Mombaça grass (Panicum maximum cv. Mombaça). In day zero had been placed 12 engorged tick females in each lot and made fertilization with urea in the dosage of 60 g in the lots of the treat group. In 27th day the grass was cutted out in 1 to 5 lots of both groups and fertilized with urea in the lots of 1 to 5 in the treat group. In 40th day was released a white flannel 1.60 x 1.00 m length on each lot to verify the presence of larvae. The results presented a difference of 85.97% (P < 0.0001) between the treat group and the control group.