Prevalence of and potential risk factors for multiple resistance to acaricides in Rhipicephalus (Boophilus) microplus ticks: A survey in the state of Rio Grande Do Sul, Brazil.

The cattle tick Rhipicephalus (Boophilus) microplus (Canestrini, 1887) (Ixodida, Ixodidae) is responsible for significant economic losses in bovine production in tropical and subtropical regions worldwide. Control of this tick predominantly involves the use of chemical acaricides; however, their indiscriminate use has led to the selection of resistant populations. A survey on tick populations was conducted in the state of Rio Grande do Sul, in Brazil, to assess the prevalence of multiple resistance to acaricides in cattle farms. Additionally, questionnaires were administered to identify potential risk factors associated with multiple resistance to acaricides. In total, 176 farms with a bovine population of ≥40 cattle were randomly assigned for tick sampling. The resistance to six acaricidal compounds was investigated by bioassays. A larval packet test was performed for amitraz, chlorpyrifos, cypermethrin, fipronil and ivermectin. Fluazuron was screened using an adult immersion test. Multiple resistance to acaricides (i.e., resistance to three or more compounds) was detected in 173 samples, representing 98% of the total samples. Among these samples, 125 (71%) showed resistance to all six compounds tested. Additionally, we classified the resistance intensity into four levels (I to IV) based on the quartile distribution of the bioassay data. Ten samples (6%) showed high and very high levels (III and IV) of resistance to all six compounds tested. Three variables were significantly associated with multiple resistance to the six acaricides tested: (i) use of injectable acaricides to control ticks, (ii) application of more than five acaricide treatments per year, and (iii) farms with larger herds (≥232 animals). These results regarding widespread resistance and the emergence of multiple resistance to acaricides ticks are alarming and highlight the significant challenge of tick control in southern Brazil.

O carrapato bovino Rhipicephalus (Boophilus) microplus (Canestrini, 1887) (Ixodida, Ixodidae) é responsável por significativas perdas econômicas na bovinocultura nas regiões tropicais e subtropicais do mundo. O controle deste carrapato é predominantemente realizado através do uso de carrapaticidas químicos; entretanto, o uso indiscriminado tem selecionado populações resistentes. Um inquérito em populações de carrapato foi conduzido no Rio Grande do Sul, Brasil, para identificar a prevalência de multirresistência a carrapaticidas em propriedades rurais. Além disso, questionários foram aplicados para determinar os potenciais fatores de risco associados à multirresistência a carrapaticidas. No total, 176 propriedades rurais com mais de ≥40 bovinos foram amostradas aleatoriamente. A resistência a seis classes de carrapaticidas foi investigada por bioensaios. O teste de pacote de larvas foi realizado para amitraz, clorpirifós, cipermetrina, fipronil e ivermectina. Fluazuron foi testado usando Teste de Imersão de carrapatos adultos. Multirresistência a carrapaticidas (resistência a três ou mais carrapaticidas) foi detectada em 173 amostras, representando 98% do total de amostras. Entre essas amostras, 125 (71%) apresentaram resistência aos seis carrapaticidas testados. Ademais, a intensidade de resistência foi classificada em quatro níveis (I a IV) baseada na distribuição de quartis dos dados dos bioensaios. Dez amostras (6%) apresentaram alto e muito alto nível de resistência (III e IV) aos seis carrapaticidas testados. Três variáveis foram significativamente associadas à multirresistência aos seis carrapaticidas testados: (i) uso de formulações injetáveis para controle de carrapato, (ii) aplicação de mais de cinco tratamentos carrapaticidas ao ano e (iii) propriedades com grande número de animais (≥232 bovinos). Esses resultados evidenciam que a disseminação da resistência e a emergência de populações de carrapatos multirresistentes é alarmante e destaca o desafio do controle de carrapatos no sul do Brasil.

RaTexT®: a novel rapid tick exposure test for detecting acaricide resistance in Rhipicephalus microplus ticks in Brazil.

BACKGROUND: Acaricide resistance in cattle ticks is a significant concern in (sub)tropical regions, particularly Brazil. The Larval Packet Test (LPT) is the standard laboratory bioassay for resistance diagnosis, which requires triplicates of seven acaricidal dilutions plus controls to cover larval mortalities ranging between 0 and 100%. The value of the LPT lies in providing resistance ratios based on the ratio between the LC50 calculated with potentially resistant and susceptible ticks. However, LC50 ratios are difficult to translate into practical advice for farmers. Moreover, LPT requires laboratory facilities to maintain susceptible tick colonies, and it takes 6 weeks to obtain the larvae to be tested by LPT derived from engorged female ticks collected from cattle in the field. Our novel approach was twofold: first, we upgraded the LPT to the Resistance Intensity Test (RIT) by adopting the latest WHO guidelines for resistance detection in mosquitoes, which combines a 1 × recommended dose with 5 × and 10 × concentrated doses to reveal low, moderate and high resistance intensity, respectively. This reduced the number of test papers and tick larvae and, more importantly, provided relevant information on the resistance level. Our second innovative step was to abolish testing larvae entirely and expose partly engorged adult ticks to the same acaricidal doses immediately after removing them from cattle in the field. This resulted in the Rapid Tick exposure Test (RaTexT®), wherein partly engorged adult ticks were exposed to an acaricide-impregnated, specially designed matrix providing test results within 24 h. This approach directly compared resistance detection in tick larvae in the RIT with resistance in adult ticks in RaTexT®. METHODS: Laboratory validation was conducted in Brazil with resistant and susceptible colonies of Rhipicephalus microplus ticks. For field validation, adult R. microplus ticks collected from different cattle farms in Brazil were evaluated for resistance to RaTexT®, and the results regarding their larval progenies were compared with those for the RIT. Partly engorged adult ticks derived from cattle infested with laboratory and field strains of R. microplus were exposed to deltamethrin in RaTexT® containers, which contained six rows of four interconnected compartments, accommodating five to eight semi-engorged female ticks with a preferred size ranging between 5 and 8 mm. The corresponding larvae of each strain were exposed in the RIT to the same deltamethrin concentrations in filter papers. RESULTS: In RaTexT®, mortality in adult ticks from a resistant strain of R. microplus from Seropédica in Brazil was 38.4%, 54.2% and 75.0% at the 1 ×, 5 × and 10 × doses of deltamethrin, respectively. In RIT, mortality of larvae from the same resistant strain was 2.0%, 4.9% and 19.5% at 1 ×, 5 × and 10 × doses, respectively. The results of RaTexT® and RIT agreed since both tests identified a high level of resistance based on a cut-off of 90% mortality. In RaTexT®, mortality of adult ticks from a susceptible strain originating from Porto Alegre was 73.8%, 92.9% and 97.6% at the 1 ×, 5 × and 10 × doses, respectively. In RIT, mortality of larvae from the susceptible strain was 95.2%, 95.2% and 96.8% at the 1 ×, 5 × and 10 × doses, respectively. Interestingly, both tests identified a low number of unexpected resistant individuals in the susceptible strain since the mortality of neither larvae nor adults reached 100%. This effect remained unnoticed in the LPT, wherein a resistance ratio of 159.5 was found based on the LC50 of the resistant strain divided by the LC50 of the susceptible strain. Next, RaTexT® was compared with RIT using adult and larval ticks derived from three field strains of R. microplus in Brazil. RaTexT® detected high levels of resistance to deltamethrin in adult ticks in all strains, which was confirmed in larvae tested by the RIT. Both tests agreed on the same resistance level with significantly lower mortality rates in larvae than in adult ticks. CONCLUSIONS: RaTexT® is a novel rapid pen-site test for detecting acaricide resistance in adult livestock ticks. It potentially replaces laborious tests using larval ticks and provides results within 24 h relevant to acaricide resistance management of livestock ticks.