Susceptibility to synthetic pyrethroids (SP´s) and the role of two major resistance mechanisms were evaluated in Mexican Rhipicephalus microplus tick populations. Larval packet test (LPT), knock-down (kdr) PCR allele-specific assay (PASA) and esterase activity assays were conducted in tick populations for cypermethrin, flumethrin and deltamethrin. Esterase activity did not have a significant correlation with SP´s resistance. However a significant correlation (p<0.01) was found between the presence of the sodium channel mutation, and resistance to SP´s as measured by PASA and LPT respectively. Just over half the populations (16/28) were cross-resistant to flumethrin, deltamethrin and cypermethrine, 21.4% of the samples (6/28) were susceptible to all of the three pyrethroids 10.7 of the samples (3/28) were resistant to flumethrin, 3.4 of the samples (1/28) were resistant to deltamethrin only and 7.1% (2/28) were resistant to flumethrin and deltamethrin. The presence of the kdr mutation correlates with resistance to the SP´s as a class. Target site insensitivity is the major mechanism of resistance to SP´s in Mexican R. microplus field strains, involving the presence of a sodium channel mutation, however, esterase-based, other mutations or combination of mechanisms can also occur.

Pyrethroid resistance in Boophilus microplus (Canestrini) (Acari: Ixodidae) was studied by correlating discriminating-dose (DD) bioassay results and esterase activity or the frequency of a sodium channel mutation known to be involved in pyrethroid resistance in nine field strains of B. microplus from Yucatan, Mexico. Two tick strains (P67 and B74) were identified as susceptible to cypermethrin, deltamethrin, and flumethrin by DD, one strain (P65) was susceptible to cypermethrin and resistant to deltamethrin and flumethrin, and six strains were resistant to cypermethrin, delta-methrin, and flumethrin (T11, M10, C54, R49, B71, and T66). By using polymerase chain reaction, only 6.0 and 6.2% of resistance allele (R) was found in the susceptible strains (P67 and B74, respectively). In the T66 strain, with 100% of larval survival to the DD of pyrethroids as measured by the larval packet test (LPT), 98.0% of the gene pool contained the R allele. Positive correlations between the larval survival and the percentage of the R allele were found (deltamethrin r2 = 0.8875, P < 0.01; cypermethrin r2 = 0.8563, P < 0.01; and flumethrin r2 = 0.8491, P < 0.01). There were no significant correlations between the level of larval survival and esterase-based hydrolytic activity. It was concluded that within the B. microplus populations studied, resistance to flumethrin, deltamethrin, or cypermethrin was because of the novel sodium channel mutation (Phe-->Ile amino acid substitution in the S6 transmembrane segment of domain III), and there was a correlation between tick mortality by pyrethroid exposure (larval survival) and the presence of R allele. It was not determined whether enhanced esterase-based hydrolytic activity was involved in pyrethroid resistance in the populations tested.

BackgroundBabesia bovis belongs to the phylum Apicomplexa and is the major causal agent of bovine babesiosis, the most important veterinary disease transmitted by arthropods. In apicomplexan parasites, the interaction between AMA1 and RON2 is necessary for the invasion process, and it is a target for vaccine development. In B. bovis, the existence of AMA1 has already been reported; however, the presence of a homolog of RON2 is unknown. The aim of this study was to characterize RON2 in B. bovis.ResultsThe B. bovis ron2 gene has a similar synteny with the orthologous gene in the B. bigemina genome. The entire ron2 gene was sequenced from different B. bovis strains showing > 99% similarity at the amino acid and nucleotide level among all the sequences obtained, including the characteristic CLAG domain for cytoadherence in the amino acid sequence, as is described in other Apicomplexa. The in silico transcription analysis showed similar levels of transcription between attenuated and virulent B. bovis strains, and expression of RON2 was confirmed by western blot in the B. bovis T3Bo virulent strain. Four conserved peptides, containing predicted B-cell epitopes in hydrophilic regions of the protein, were designed and chemically synthesized. The humoral immune response generated by the synthetic peptides was characterized in bovines, showing that anti-RON2 antibodies against peptides recognized intraerythrocytic merozoites of B. bovis. Only peptides P2 and P3 generated partially neutralizing antibodies that had an inhibitory effect of 28.10% and 21.42%, respectively, on the invasion process of B. bovis in bovine erythrocytes. Consistently, this effect is additive since inhibition increased to 42.09% when the antibodies were evaluated together. Finally, P2 and P3 peptides were also recognized by 83.33% and 87.77%, respectively, of naturally infected cattle from endemic areas.ConclusionsThe data support RON2 as a novel B. bovis vaccine candidate antigen that contains conserved B-cell epitopes that elicit partially neutralizing antibodies.Electronic supplementary materialThe online version of this article (10.1186/s13071-018-3164-2) contains supplementary material, which is available to authorized users.

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