Microbial agents for the control of ticks Rhipicephalus microplus.

Ticks are ectoparasites responsible for the transmission of various pathogens to vertebrates. They represent one of the major threats to livestock production worldwide due to their impact on the health, production and welfare of livestock destined for human consumption. The development of resistance to the main families of ixodicides used for their control has led to the search for new alternatives, where microbial control is an option. The use of microbial control agents against the tick Rhipicephalus microplus is reviewed in this paper. Bacteria such as Bacillus thuringiensis, Serratia marcescens and Staphylococcus spp. the nematodes Steinernema spp. and Heterorhabditis spp. as well as the fungi Metarhizium anisopliae and Beauveria bassiana are the most studied organisms for use as biological control agents against ticks. Laboratory, stable and field trials with free-living and parasitised ticks have shown that microbial agents can control both susceptible and ixodicide-resistant tick populations. However, multidisciplinary studies using novel tools like genomics, transcriptomics and proteomics should be carried out to understand the virulence factors which microbial agents use to induce pathogenesis and virulence in ticks. In addition, applied research will be carried out with the aim of improving techniques for large-scale application, as well as the improvement of cultivation, storage, formulation and application methods.

Testing Efficacy of a Conserved Polypeptide from the Bm86 Protein against Rhipicephalus microplus in the Mexican Tropics.

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.

Histometric and morphological damage caused by Serratia marcescens to the tick Rhipicephalus microplus (Acari: Ixodidae).

Rhipicephalus microplus tick is the ectoparasite causing the greatest economic losses in the livestock industry. Multi-resistance in ticks is increasing, generating the inefficiency of traditional ixodicides, for which biological control has been proposed as an alternative. In this work, we analyze the histomorphological damage caused by the bacterial strain EC-35 on Rhipicephalus microplus. The ixodicidal effect of EC-35 total protein was evaluated on larval or adult ticks comparing with the commercial ixodicide coumaphos 0.02% as a control. Female ticks were processed using the paraffin-embedding technique and stained with hematoxylin-eosin. Also, the pathogenicity of EC-35 was evaluated by capillary feeding and coelom inoculation tests. The identification of the bacterium was performed using the molecular markers 16S RNA and rpoB, by PCR and sequencing technique, and the evolutionary distance was analyzed by Bayesian phylogenetic inference. No differences were observed in the perimeter and area of larvae treated with EC-35 or Coumaphos. The thickness of the integument decreased a 65% with the EC-35 treatment (6.01 ± 0.6 µm) and of 30% in coumaphos (12.04 ± 1.2 µm) in larvae compared with the control group (18.41 ± 2 µm), while no difference was found in adult ticks. The capillary feeding test and coelom inoculation with EC-35 showed an inhibition of reproductive potential of 99.8 ± 7 and an oviposition Inhibition 97 ± 3.02%. The EC-35 strain was genetically related to Serratia marcescens, concluding that these bacteria caused high mortality, oviposition Inhibition, and integument thinning and drastic loss of histoarchitecture in R. microplus tick larvae.

Experimental vaccination in rabbits using the peptide RmS-17 antigen reduces the performance of a Mexican Rhipicephalus microplus tick strain.

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.

Characterization of a strain of Serratia sp. with ixodicide activity against the cattle tick Rhipicephalus microplus.

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.

Genetic diversity of the ATAQ gene in Rhipicephalus microplus collected in Mexico and implications as anti-tick vaccine.

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.