RaTexT®: a novel Rapid Tick exposure Test for detecting acaricide resistance in livestock ticks in Brazil

Abstract

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 six 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 1x recommended dose with 5x and 10x concentrated doses to reveal low, moderate, and high resistance intensity, respectively. This reduced the number of test papers and tick larvae but, 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 hours. 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 in RaTexT^® and compared with results with the RIT on their larval progenies. 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 impregnated 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 1x, 5x and 10x doses of deltamethrin respectively. In RIT, mortality of larvae from the same resistant strain was 2.0%, 4.9% and 19.5 % at 1x, 5x and 10x 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 1x, 5x and 10x doses, respectively. In RIT, mortality of larvae from the susceptible strain was 95.2%, 95.2% and 96.8 % at the 1x, 5x and 10x 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, which determined a resistance ratio of 159.5. 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 hours relevant to acaricide resistance management of livestock ticks.