RESUMO

Rhipicephalus sanguineus s.l. is an intra- and peridomiciliary tick of medical and veterinary importance, whose control is carried out through the application of various ixodicides, to which it can develop resistance. The objective of this work was to determine the discriminant doses (d.d.) to identify resistance against three ixodicides - fipronil, flumethrin and coumaphos - in R. sanguineus s.l. larvae originating from Mexico. Engorged ticks were collected from naturally infested dogs residing in rural communities in five Mexican states; the dogs had no history of recent treatment with ixodicides. All ticks were identified as R. sanguineus s.l. They were incubated for 25 days and their progeny was used in the larval package test (LPT) to be exposed to d.d. as determined in Rhipicephalus microplus, and those that were susceptible were analyzed using the LPT with six concentrations, for fipronil from 0.1 to 0.003125%, for flumethrin from 0.02 to 0.000625% and for coumaphos from 0.4 to 0.0125%. Mortality was analyzed with Probit methodology, to calculate the 50 and 99% lethal concentrations (LC50 and LC99). The d.d. was determined as a consensus value by multiplying the LC99 value × 2. Nine double susceptible samples (fipronil + flumethrin) and one triple susceptible sample (fipronil + flumethrin + coumaphos) were found; the d.d. determined were fipronil 0.05%, flumethrin 0.01% and coumaphos 0.43%. These doses can be used to rapidly and inexpensively identify resistant populations in samples collected in the field.

RESUMO

The brown dog tick, Rhipicephalus sanguineus s.l., is considered the most widely distributed three-host tick in the world and has medical and veterinary importance; the control of infestation is carried out with acaricides, towards which it can develop resistance. This study aimed to determine the discriminant dose (d.d) of amitraz to identify resistance in R. sanguineus s.l. larvae natives from Mexico and to evaluate its application in field-collected ticks. Engorged ticks were collected from naturally infested dogs residing in rural communities and were incubated for 25 days, and their progeny was used in a larval immersion test (LIT) to be exposed to the d.d. determined in Rhipicephalus microplus, and those that were susceptible were analyzed using the LIT in six concentrations. Mortality was analyzed through probit methodology to calculate the lethal concentration (LC) 50 and 99. The d.d. was determined as a consensus value by multiplying the LC99 × 2, and then, we proceeded to evaluate it in in-field samples by using the LIT technique. The d.d. calculated was 4 ppm. The in-field evaluation found 64% of the resistant samples to amitraz with mortality percentages between 98.3% and 0.35%. This dose can be used to rapidly and inexpensively identify resistant populations in samples collected in the field.

RESUMO

Rhipicephalus microplus economically impacts cattle production in tropical and subtropical countries. Application of acaricides constitutes the major control method; however, inadequate use has increased resistant tick populations, resulting in environmental and cattle product contamination. Anti-tick vaccines based on the Bm86 antigen are an environmentally friendly, safe, and economically sustainable alternative for controlling R. microplus infestations. Nevertheless, variable efficacy has been experienced against different geographic tick strains. Herein, we evaluated the efficacy of a conserved polypeptide Bm86 derived from a Mexican R. microplus strain previously characterized. Twelve cows were assigned to three experimental groups and immunized with three doses of the polypeptide Bm86 (pBm86), adjuvant/saline alone, and Bm86 antigen (control +), respectively. Specific IgG antibody levels were measured by ELISA and confirmed by Western blot. In addition, the reproductive performance of naturally infested R. microplus was also determined. The more affected parameter was the adult female tick number, with a reduction of 44% by the pBm86 compared to the controls (p < 0.05), showing a vaccine efficacy of 58%. Anti-pBm86 IgG antibodies were immunogenic and capable of recognizing the native Bm86 protein in the eggs, larvae, and guts of R. microplus. The negative correlation between antibody levels and the reduction of naturally tick-infested cattle suggested that the effect of the polypeptide Bm86 was attributed to the antibody response in immunized cattle. In conclusion, the polypeptide Bm86 showed a specific immune response in cattle and conferred protection against R. microplus in a Mexican tropical region. These findings support further experiments with this antigen to demonstrate its effectiveness as a regional vaccine.

RESUMO

The tick vector Rhipicephalus microplus is considered one of the main problems in cattle production in tropical and subtropical regions. Anti-tick vaccines may form an alternative tick control method to the use of acaricides, and tick salivary proteins, such as Serpins, may be valuable as target antigens for developing anti-tick vaccines. In this study, we synthesized a recombinant peptide derived from Serpin RmS-17 protein using an Escherichia coli expression system and characterized the efficacy of the peptide RmS-17 for the control of R. microplus females infesting rabbits. Twelve New Zealand white rabbits were assigned to three experimental groups and vaccinated with three subcutaneous doses of the peptide RmS-17, recombinant R. microplus Bm86 antigen, and adjuvant/saline alone. The tick challenge was conducted with 120 R. microplus adults (60 females and 60 males) per animal, with the ticks placed inside a cotton sleeve glued to the back of the rabbit. Serum antibody levels (IgG) were assessed by ELISA and confirmed by Western blot; also, the reproductive performance of R. microplus was determined. The results showed that experimental vaccination in rabbits using the peptide RmS-17 antigen had a vaccine efficacy of 79% based on reductions in adult tick number, oviposition, and egg fertility compared to control animals. The peptide RmS-17 vaccinated rabbits developed a strong humoral immune response expressed by high anti-pRmS-17 IgG levels, and the Western blot analysis confirmed that it is immunogenic. The efficacy for the Bm86 vaccine was 62%, which is within the range of efficacy reported previously for Bm86 vaccine. The negative correlation between antibody levels and reduction in tick number strongly suggests that the effect of the vaccine was the result of the antibody response in vaccinated rabbits. In conclusion, this is the first study to evaluate the efficacy of the peptide RmS-17 against R. microplus tick infestation and show it to be immunogenic and protective in a rabbit model.

RESUMO

The cattle tick Rhipicephalus microplus affect animal health, welfare, and cattle production in tropical and subtropical zones of the world. Anti-tick vaccines have been an effective alternative for cattle tick control instead of traditional chemical products. To date, Subolesin antigen has shown efficacy for the control of tick infestation in cattle, and previous studies showed that one peptide derived from this protein has demonstrated to elicit a strong and specific humoral immune response. Based on these findings, herein we characterized the efficacy of the peptide Subolesin for the control of cattle tick, R. microplus infestation under field conditions. Twenty-four female calves were assigned to four experimental groups and immunized with three subcutaneous doses of the peptide Subolesin, Bm86, both antigens (dual vaccine) and adjuvant/saline alone, respectively. Serum antibody levels (IgG) were assessed by ELISA and confirmed by Western blot; also, reproductive performance of naturally infested R. microplus was determined. The results showed that immunizations with the experimental antigens reduced tick infestations with vaccine's efficacy of 67 % (peptide Subolesin), 56 % (Bm86), and 49 % (dual vaccine) based on adult tick numbers, oviposition, and egg fertility between vaccinated and control animals. Peptide Subolesin-immunized calves developed a strong humoral immune response expressed by high anti-pSubolesin IgG levels, and the Western blot analysis confirmed that it is immunogenic. Cattle receiving Bm86 and dual vaccine showed less protection, although Bm86 was within the range reported previously. The negative correlation between antibody levels and reduction of naturally infested R. microplus strongly suggested that the effect of the vaccine was the result of the antibody response in immunized cattle. In conclusion, it was demonstrated that the peptide Subolesin induced a specific immune response in cattle under field conditions, resulting in reduced R. microplus populations in subsequent generations. Finally, integrated tick control must consider anti-tick vaccines as a cost-effective, sustainable, and successful tool for controlling cattle tick infestations.

Assuntos

RESUMO

Cattle ticks are considered the most important ectoparasite in the livestock industry. Rhipicephalus microplus causes economic losses both through direct feeding on livestock and through disease transmission. Reports of the failure of chemical ixodicides to control this tick have led to a search for control alternatives, such as bacteria with ixodicide activity. The objective of this work was to select a bacterial strain with ixodicide activity against R. microplus. In total, 83 bacterial strains were isolated from soil and dead R. microplus specimens, and all strains were evaluated against larvae in a screening test. Bacteria with ixodicide activity were evaluated in larvae and engorged adult female ticks. The larvae were challenged using the larval immersion test (LIT) with 20 µg/mL total protein. The median lethal concentration (LC50) for larvae was obtained by using nine total protein concentrations. Engorged adult female ticks were challenged using the adult immersion test (AIT) with six protein concentrations. We evaluated adult mortality on day 10, oviposition rate on day 14 and hatching rate on day 40 after challenge. Only one bacterial strain (EC-35) showed ixodicide activity against larvae and adult R. microplus. The highest larval mortality, 52.3%, occurred with a total protein concentration of 40 µg/mL, and the LC50 was 13.9 µg/mL of protein. In adults, a total protein concentration of 10 µg/mL had the highest mortality (55%), oviposition inhibition (50.9%) and reproductive potential inhibition (52.5%). However, there was no significant effect on hatching. The 16S rRNA gene sequence showed 99% identity of EC-35 with Serratia sp.

Assuntos

RESUMO

The cattle tick Rhipicephalus microplus has a large impact on cattle production due to its bloodsucking habit and transmission of pathogens that cause babesiosis and anaplasmosis. Application of acaricides constitutes the major control method but is often accompanied by serious drawbacks, including environmental contamination and an increase in acaricide resistance by ticks. The recent development of anti-tick vaccines has provided positive results in the post-genomic era, owing to the rise of reverse vaccinological and bioinformatics approaches to analyze and identify candidate protective antigens for use against ticks. The ATAQ protein is considered a novel antigen for the control of the cattle tick R. microplus; it is expressed in midguts and Malpighian tubules of all ticks from the Rhipicephalus genus. However, genetic diversity studies are required. Here, the ATAQ gene was sequenced of seven R. microplus tick isolates from different regions in Mexico to understand the genetic diversity. The results showed that sequence identity among the Mexican isolates ranged between 98 and 100% and 97.8-100% at the nucleotide and protein levels, respectively. Alignments of deduced amino acid sequences from different R. microplus ATAQ isolates in Mexico revealed a high degree of conservation. However, the Mexican isolates differed from the R. microplus "Mozambique" strain, at 20 amino acid residues. Finally, the analysis of more R. microplus isolates, and possibly of other Rhipicephalus species, to determine the genetic diversity in the ATAQ locus is essential to suggest this antigen as a vaccine candidate that might control tick infestations.

Assuntos

RESUMO

Rhipicephalus microplus (formerly Boophilus microplus) ticks are potential vectors of several pathogens of livestock especially in tropical and subtropical regions where may have substantial effects on economic development. Among tick-borne pathogens, Anaplasma marginale is considered one of the most important in domestic and wild ruminants worldwide. Different molecular mechanisms have been employed by both ticks and these intracellular pathogens, in order to be able to adapt and survive. Subolesin, originally called 4D8, is an evolutionarily well-preserved protein among ixodid tick species. This new antigen was found to be protective against tick infestations when used as a vaccine, as it has an essential role in tick blood digestion, development and infection of host cells by A. marginale. Recent studies have demonstrated that infection of both tick and vertebrate host cells with this microorganism changed gene expression. Therefore, the main objective of this study was to investigate subolesin expression in uninfected and A. marginale-infected R. microplus salivary glands by real-time reverse transcriptase (RT)-PCR. To analyze the differential expression of the recombinant protein subolesin, the gene was previously expressed from ticks infected with A. marginale. Results from this study revealed that, the expression of subolesin was significantly higher in salivary glands of infected R. microplus in comparison to uninfected ones.

Assuntos