Evaluation of the impact of successive acaricide treatments on resistance evolution in Rhipicephalus microplus populations: Monodrugs versus drug combinations.

The aim of this work was to comparatively evaluate the evolution of resistance in Rhipicephalus microplus tick populations exposed to successive treatments with monodrug-based formulations and combinations of them in the same commercial formulation. Thirty-six heifers, naturally infested with R. microplus, were divided into three groups (G) and subjected to three successive treatments, on days 0 (Nov-2021), 43 (Jan-2022) and 78 (Feb-2022), with the following formulations: I) ivermectin 3.15% (Ivomec Gold®) (GI), II) fipronil 1% (Ectoline®) (GII) and III) a combination of fipronil 2% and ivermectin 1% (Vaquero®) (GIII). From Nov-2021 to Dec-2022, counts of R. microplus were periodically performed to evaluate the tick infestation. Engorged females were collected at the beginning and end of the trial to determine the evolution of tick resistance to ivermectin and fipronil by in vitro bioassays. At the end of trial, GII and GIII had higher tick counts (39.18 ± 11.88 and 38.33 ± 14.31, respectively) than group I (5.11 ± 6.24) (P<0.05). The in vitro results shows that the resistance ratio (RR50) values after the treatments were higher for fipronil (5.584 and 5.649 for GII and GIII, respectively) than for ivermectin (1.165 and 1.088 for GI and GIII, respectively). In the group treated with the combination (GIII), the RR50 increased for both drugs simultaneously. These results suggest that the successive use of drug combinations could exacerbate the problem of multi-resistance of R. microplus to chemical acaricides.

A comparison of two in vitro bioassays to detect resistance of the cattle tick Rhipicephalus microplus to fipronil.

The aim of this work is to compare the sensitivity of two in vitro bioassays to detect resistant to fipronil in Argentinean populations of the cattle tick Rhipicephalus microplus. Two different larval bioassays prepared with technical grade (97%) fipronil were compared: larval immersion test (LIT) and larval packet test (LPT). Seven strains from different provinces were treated with both assays. Colonia Tabay, Colonia Benítez, Intiyaco and Quimili strains were considered resistant in both LIT and LPT bioassays. The 95% confidence intervals (IC95) for lethal concentration 50% (LC50) did not overlap with the susceptible reference strain (SRS) and all the values of RR50 obtained were higher than 2. Garabato and Federal strains were considered as susceptible for both techniques because the IC95 for the LC50 overlapped with those of the SRS and the RR50 values were lesser than 2. An ambiguous situation occurs with Reconquista strain. This strain was considered as susceptible with LPT and with incipient resistant after LIT trial. The analysis of the results indicates that both LIT and LPT trials have enough sensibility to differentiate resistant and susceptible strains, but LIT was more sensitive than LPT when the resistance is incipient.

Two novel Ehrlichia (Rickettsiales: Anaplasmataceae) strains detected in ticks (Ixodida, Ixodidae) and opossums (Didelphimorphia: Didelphidae) in Argentina.

The aim of this study is to determine if there is circulation of microorganisms of the genus Ehrlichia in opossums Didelphis albiventris and their ticks from the Humid Chaco in Argentina. Blood samples of 15 specimens of the opossum D. albiventris were analysed. Immature stages of the ticks Amblyomma ovale (Larvae=26; Nymphs=10), Amblyomma sculptum (Larvae=86; Nymphs=6) and Ornithodoros sp. cf. O. mimon (Larvae=90) were also analyzed. DNA was extracted individually from blood samples and ticks. Molecular detection of Ehrlichia agents was performed targeting two different loci: 16S rRNA and dsb gen. The phylogenetic analyses showed that the Ehrlichia sp. detected in D. albiventris in this study is identical to Ehrlichia sp. strain Natal previously detected in two marsupials from Brazil. Furthermore, a new Ehrlichia strain was amplified from an A. ovale nymph (named as Ehrlichia sp. strain El Bagual) which is phylogenetically closely related to a strain of Ehrlichia sp. detected in Bradypus tridactylus in Brazil. The findings of the current study represent the first report of these two strains of Ehrlichia for Argentina, showing that the diversity of Ehrlichia spp. is greater than previously assumed. Further studies should determine the epidemiological relevance of these findings.

Analysis of sequence variations in the GABA gated chloride gene associated with resistance to fipronil in Rhipicephalus (Boophilus) microplus ticks from Argentina.

This study aimed to evaluate the sequence variations of the GABA-Cl gene of Rhipicephalus (Boophilus) microplus ticks from Argentina with different resistance levels to fipronil. Genomic DNA was obtained from engorged females (n = 50) of fipronil-susceptible and resistant tick field populations from five localities in northern Argentina. Tick populations were tested for fipronil resistance by in vitro larval packet test. DNA encoding amino acid residues 264-360 of the GABA-Cl was amplified by PCR. PCR products were sequenced and the translated amino acid sequences were aligned and compared among each other and with sequences of the GABA-Cl belonging to the Brazilian and Uruguayan reference strains fipronil-susceptible (Mozo) and resistant (RFSan and Juarez). All the GABA-Cl amino acid sequences obtained from the R. microplus specimens from Argentina were identical, regardless of the fipronil resistance-susceptibility status of each population. Mutations present in GABA-Cl sequence of RFSan and Juarez strains were not present in any of the populations from Argentina analyzed. These results indicate the failure of this gene as a molecular marker of resistance to fipronil, at least in the R. microplus populations from northern Argentina.

Resistance of the cattle tick Rhipicephalus (Boophilus) microplus to fluazuron in Argentina.

The aim of this work is to report the presence of resistance to fluazuron in a population of Rhipicephalus microplus in Argentina. The evidence was obtained from field and in vitro trials. In the field trial, cattle infested with ticks was treated with two commercial formulations of fluazuron. The in vitro trial (adult immersion test, AIT) was performed by using technical grade fluazuron. In the field trial, there were no significant differences between the treated and control groups between days 2 and 34 post-treatment. The only exceptions (treated group I in day 14 post-treatment, treated group II in days 23 and 29 post-treatment) had a significantly lower tick load than the untreated group, but the efficacy was not higher than 70%. Viable engorged females were collected on both groups of treated bovines in all counts, and the production of viable larvae was not precluded with the application of the two commercial formulations of fluazuron evaluated in this study. The results obtained with the in vitro assay (AIT) also indicate that the R. microplus population tested in this work has a higher level of resistance to fluazuron than another susceptible field strain. The integrated analysis of the field and in vitro trials clearly reveals the emergence of resistance to fluazuron in a R. microplus population from Argentina. This diagnosis of resistance does not imply that the fluazuron has lost its functionality at a regional scale, but it highlights the need to establish control strategies that minimize the use of this drug in order to preserve its functionality as an acaricide.

In vitro diagnosis of resistance of the cattle tick Rhipicephalus (Boophilus) microplus to fipronil in Argentina.

The aim of this work was to adapt and apply the in vitro larval bioassay packet test (LPT) to diagnose resistance of Rhipicephalus microplus ticks to fipronil in Argentina. Ten serial dilutions of fipronil were included in the LPT bioassay. Seven populations of R. microplus from six Argentinean Provinces were evaluated. Samples from Colonia Benítez, Quimilí and Intiyaco were unambiguously classified in the category 'resistant'. In all these cases the 95% confidence intervals did not overlap with that of the susceptible reference strain and the slope values were lower than the slope value obtained for the susceptible reference strain. The resistance ratio (RR50) values of the populations from Colonia Benítez I and II, Quimilí and Intiyaco were 2.23, 3.49, 6.84 and 3.39, respectively. Populations from El Colorado I and II, Acambuco, Vera and Feliciano were classified as 'susceptible' because the 95% confidence intervals overlapped with that of the susceptible reference strain and the RR50 values never exceeded 1.32. The results of these LPT bioassays indicate the presence of fipronil resistant R. microplus populations in Argentina.

Resistance of the cattle tick Rhipicephalus (Boophilus) microplus to ivermectin in Argentina.

Resistance to ivermectin in populations of the cattle tick Rhipicephalus microplus in Argentina was diagnosed in this work. The in vitro larval immersion test (LIT) was used to determine quantitatively the levels of resistance to ivermectin in different populations of R. microplus. Additionally, field trials to control natural infestations of R. microplus on cattle with a commercial formulation of ivermectin 3.15% were carried and jointly analyzed with the in vitro assays. The phenotypic response of the populations analyzed was not uniform. Five of them were classified as susceptible, four populations as resistant, and one in the category "incipient resistance". Regarding the field trials, the therapeutic efficacy in a population classified with LIT as susceptible achieved values higher than 94% two weeks after treatment, and no reproductively viable females were observed after the second day post-treatment. Conversely, the values of efficacy percentage in a population (named as "San Martín") classified with LIT in the category "incipient resistance" never exceeded the 70.8%, and engorged females were collected in practically all counts. The population "San Martín" was classified in the category "incipient resistant" with LIT analysis, but the field trial unambiguously shows that this tick population is resistant. The comparison of the results obtained with LIT in vitro assays and through field trials shows that biased estimations of resistance levels may occur when resistance ratios (RR) values are ≤2, and additional field efficacy trials could be needed to know with precision the status of the tick populations evaluated.