Oral vaccine formulation combining tick Subolesin with heat inactivated mycobacteria provides control of cross-species cattle tick infestations

Tick vaccines are necessary as part of a One Health approach for the control of tick infestations and tick-borne diseases. Subolesin (SUB, also known as 4D8) is a tick protective antigen that has shown efficacy in vaccine formulations for the control of ectoparasite infestations and pathogen infection/transmission. A recent proof-of-concept study reported oral vaccination combining Rhipicephalus microplus SUB with heat inactivated Mycobacterium bovis (IV) as an immunostimulant for the control of cattle tick infestations. Based on the efficacy of Rhipicephalus decoloratus SUB for the control of multiple cattle tick species in Uganda, herein we design a controlled pen trial using an oral formulation combining R. decoloratus SUB with IV for the control of R. decoloratus and Rhipicephalus appendiculatus cattle tick infestations. Vaccine efficacy (E) of SUB + IV on tick life cycle was compared with IV and SUB alone and with PBS as control. The IgG antibody titers against SUB and M. bovis P22 and the serum levels of selected protein immune biomarkers (IL-1beta, TNF-alpha, C3) were determined and analyzed as possible correlates of protection. Oral immunization with IV and SUB alone and in SUB + IV combination were effective for the control of tick infestations (E = 71–96% for R. decoloratus and 87–99% for R. appendiculatus) with highest E (higher than 95%) for SUB + IV. The results demonstrated that oral immunization with the SUB + IV formulation resulted in effective control of cattle tick infestations through the activation of multiple immune mechanisms. These results support the application of oral vaccine formulations with SUB + IV for the control of cattle infestations with Rhipicephalus species towards improving animal health.     

Tick species infesting cattle in the central region of Burkina Faso: Presence of Rhipicephalus microplus less than ten years after its first identification in the Southwest part of the country

Ticks are among the most important vectors for medical and veterinary importance in African countries, representing thus an obstacle for development and improvement of livestock production. The central part of Burkina Faso is an arid region which has a low diversity of tick species and in which Rhipicephalus microplus has been absent. This study aimed to examine ticks infesting cattle within this region, focusing on the peripheral area of Ouagadougou. Tick sampling was undertaken monthly on 30 selected cattle in the state breeding station of Loumbila, from June 2017 to February 2018. Morphological identification was carried out based on previous established keys and catalogue. A total of 1,076 ticks belonging to four genera and seven species were collected. Amblyomma variegatum (50.5%) was the most abundant species followed by Hyalomma rufipes (27.4%), Hyalomma truncatum (13.7%), Rhipicephalus decoloratus (4.5%), Hyalomma impressum (2.4%), Rhipicephalus microplus (1.5%), and Rhipicephalus sanguineus sensu lato (0.1%). The results suggested a decrease of A. variegatum and Hyalomma spp. load compared to data collected in 1996 from the same study area.Presence of R. microplus and R. decoloratus has been highlighted in the studied area for the first time. This finding highlights the relevance of regular investigations on tick species infesting livestock utilizing collaboration with different stakeholders.     

Immunization of cattle with Ra86 impedes Rhipicephalus appendiculatus nymphal-to-adult molting

Commercial vaccines based on the tick gut protein Bm86 have been successful in controlling the one-host tick Rhipicephalus (Boophilus) microplus and provide heterologous protection against certain other non-target ixodid tick species. This cross protection, however, does not extend to the three-host tick R. appendiculatus, the vector of the protozoan parasite Theileria parva. When transmitted to cattle, T. parva causes the often fatal disease East Coast fever. Here, we used insect cell-expressed recombinant versions of the R. appendiculatus homologs of Bm86, named Ra86, to vaccinate cattle. We measured multiple fitness characteristics for ticks that were fed on cattle Ra86-vaccinated or unvaccinated. The Ra86 vaccination of cattle significantly decreased the molting success of nymphal ticks to the adult stage. Modeling simulations based on our empirical data suggest that repeated vaccinations using Ra86 could reduce tick populations over successive generations. Vaccination with Ra86 could thus form a component of integrated control strategies for R. appendiculatus leading to a reduction in use of environmentally damaging acaricides.     

Identification of a synthetic peptide inducing cross-reactive antibodies binding to Rhipicephalus (Boophilus) decoloratus, Rhipicephalus (Boophilus) microplus, Hyalomma anatolicum anatolicum and Rhipicephalus appendiculatus BM86 homologues

The BM86 antigen, originally identified in Rhipicephalus (Boophilus) microplus, is the basis of the only commercialized anti-tick vaccine. The long-term goal of our study is to improve BM86 based vaccines by induction of high levels of tick gut binding antibodies that are also cross-reactive with a range of BM86 homologues expressed in other important tick species. Here we have used a BD86 derived synthetic peptide, BD86-3, to raise a series of mouse monoclonal antibodies. One of these mAbs, named 12.1, recognized BM86 homologues in immuno-histochemical analyses in four out of five tick species including R. (B.) microplus, Rhipicephalus (Boophilus) decoloratus, Hyalomma anatolicum anatolicum and Rhipicephalus appendiculatus. Our results indicate that broadly cross-reactive tick gut binding antibodies can be induced after immunization with a synthetic peptide derived from the protein BD86.     

Characterization of ferritin 2 for the control of tick infestations

Ixodes ricinus is one the most abundant tick species in Europe and these ticks transmit pathogens causing human and animal diseases. The cattle ticks, Rhipicephalus (Boophilus) spp., affect cattle production in tropical and subtropical regions of the world. Development of vaccines directed against tick proteins may reduce tick infestations and the transmission of tick-borne pathogens. However, a limiting step in tick vaccine development has been the identification of tick protective antigens. Herein, the tick iron metabolism pathway was targeted in an effort to identify new tick protective antigens. Recombinant I. ricinus (IrFER2) and Rhipicephalus microplus (RmFER2) ferritin 2 proteins were expressed in Escherichia coli and used to immunize rabbits and cattle, respectively. Vaccination with IrFER2 reduced I. ricinus tick numbers, weight and fertility in rabbits with an overall vaccine efficacy (E) of 98%. Control of cattle tick, R. microplus and Rhipicephalus annulatus infestations was obtained in vaccinated cattle with overall E of 64% and 72%, respectively. Notably, the efficacy of the RmFER2 vaccine was similar to that obtained with Bm86 against R. microplus. These collective results demonstrated the feasibility of using ferritin 2 to develop vaccines for the control of tick infestations.     

Lesser protein degradation machinery correlates with higher BM86 tick vaccine efficacy in Rhipicephalus annulatus when compared to Rhipicephalus microplus

Infestations with cattle ticks, Rhipicephalus (Boophilus) microplus and Rhipicephalus annulatus, economically impact cattle production in tropical and subtropical regions of the world. Vaccines containing the recombinant R. microplus BM86 gut antigen were developed and commercialized to induce an immunological protection in cattle against tick infestations. These vaccines demonstrated that tick control by vaccination is cost-effective, reduces environmental contamination and prevents the selection of drug resistant ticks that result from repeated acaricide applications. The protection elicited by BM86-containing vaccines against tick infestations is mediated by a collaborative action between the complement system and IgG antibodies. The efficacy of the vaccination with BM86 and other tick antigens is always higher for R. annulatus than against R. microplus, suggesting that tick genetic and/or physiological factors may affect tick vaccine efficacy. These factors may be related to BM86 protein levels or tick physiological processes such as feeding and protein degradation that could result in more efficient antibody–antigen interactions and vaccine efficacy. To test this hypothesis, we compared the proteome in R. annulatus and R. microplus female ticks after feeding on BM86-vaccinated and control cattle. The results showed that cattle proteins were under represented in R. annulatus when compared to R. microplus, suggesting that R. annulatus ticks ingested less blood, a difference that increased when feeding on vaccinated cattle, probably reflecting the effect of antibody–BM86 interactions on this process. The results also showed that tick protein degradation machinery was under represented in R. annulatus when compared to R. microplus. BM86 mRNA and protein levels were similar in both tick species, suggesting that lesser protease activity in R. annulatus results in more efficient antibody–antigen interactions and higher vaccine efficacy. These results have important implications for tick vaccine research, indicating that not only genetic differences, but also physiological factors may influence tick vaccine efficacy.     

Identification of Rickettsia spp., Anaplasma spp., and an Ehrlichia canis-like agent in Rhipicephalus microplus from Southwest and South-Central China

Rhipicephalus microplus is considered to be the most important tick infesting cattle, buffalo, horse, goats as well as other animals. They transmit diseases between domestic animals and act as vectors of a variety of zoonotic pathogens. Although pathogens harbored by R. microplus have been extensively studied, the Rickettsiales pathogens vectored by R. microplus in some areas of China remained largely unexplored. From August to October 2020, a total of 291 R. microplus ticks were collected from goats and cattle in three Southern China provinces, Guangxi (n = 138), Sichuan (n = 120) and Hubei (n = 33) provinces. Phylogenetic analysis based on COI gene sequences shows that these ticks are divided into three distinct clades, indicating the remarkable genetic diversity of R. microplus ticks in China. These samples were subsequently screened for the presence of Rickettsia, Anaplasma and Ehrlichia using conventional PCR and sequencing. Subsequently, five bacterial species were identified. Out of the 120 tick DNA samples from Sichuan province, 35.83% (43/120) were positive for Rickettsia sp. belonging to spotted fever group (SFG), 12.50% (15/120) were positive for Anaplasma marginale and 0.83% (1/120) was identified as A. platys. From the 138 DNA samples from Guangxi province, an Ehrlichia canis-like and Rickettsia sp. were detected, with a positive rate of 11.59% (16/138) and 2.17% (3/138), respectively. A. capra DNA was detected in 4 out of 33 (12.12%) samples from Hubei province. Notably, the 16S, gltA and groEL sequences of the E. canis-like are closely related to the E. canis strain previously identified from China, and form a distinct cluster in the phylogenetic trees. Collectively, our results expand the knowledge on tick-borne Rickettsiales pathogens in China. Because the state of engorgement of ticks was not recorded, it is not clear at this stage whether these pathogens are infecting the ticks or are simply present in the blood meal. Given the public health significance of SFG Rickettsia, A. capra, A. platys and E. canis, a thorough investigation of the diversity and presence of pathogens in R. microplus in areas with tick-associated diseases are needed.     

Vaccine efficacy of recombinant BmVDAC on Rhipicephalus microplus fed on Babesia bigemina-infected and uninfected cattle

Rhipicephalus microplus is the most widely distributed tick worldwide and causes significant economic losses in the livestock industry. It directly affects hosts (especially in large infestations) by feeding on blood and piercing the skin and indirectly affects hosts as a vector of pathogens that cause infectious diseases, such as bovine babesiosis. Current research on the control of ticks is focused on integrated tick control programmes, including vaccination treatment with acaricides and completely blocking pathogen transmission. Our previous studies showed that R. microplus VDAC (BmVDAC) expression is modulated by Babesia bigemina infection. VDAC is a mitochondrial protein with multiple functions in addition to its primary role as a central component of the apoptotic machinery. In this paper, we evaluated BmVDAC as an anti-tick vaccine and its capacity to block the infection of Babesia bigemina in ticks. Our results demonstrate that rBmVDAC is immunogenic and that antibodies specifically recognize the native protein from midguts of R. microplus. Immunization with rBmVDAC afforded an 82% efficacy against R. microplus infestation in the group of vaccinated cattle compared with the control group. In contrast, rBmVDAC showed a lower efficacy of 34% against tick infestation in cattle vaccinated with rBmVDAC, infested with R. microplus and infected with B. bigemina. The main effect on ticks fed in vaccinated and infected cattle was a 34% reduction in egg fertility (DF) compared to ticks fed on the control group. There was no reduction in the B. bigemina parasite levels of ticks fed on rBmVDAC-vaccinated cattle. These results suggest that the rBmVDAC protein could be tested as a vaccine for the control of tick infestation.     

Successive treatments with ivermectin (3.15%) to control the tick Rhipicephalus (Boophilus) microplus in cattle: Pharmacokinetic and efficacy assessment

This study aimed to evaluate the pharmacokinetics, the potential accumulation in the body of treated animals and the efficacy of ivermectin long-acting formulation (3.15%) against the cattle tick Rhipicephalus (Boophilus) microplus in a scheme of three successive treatments. Fifteen 12-month-old heifers, naturally infested with R. microplus, were divided into two groups (G). Cattle from GI (n = 10) were subjected to three treatments with ivermectin 3.15% (IVOMEC GOLD®, Merial Argentina S.A.) at a rate of 1 mL/50 kg on days 0, 35, and 70. Cattle from GII (n = 5) were not treated. From day 1 to 202 post-treatment blood samples were taken to measure ivermectin concentrations by HPLC and female ticks (4.5–8 mm) were counted to evaluate the efficacy of the treatment. The level of tick resistance to ivermectin was evaluated before and after finishing the scheme of successive treatments by larval immersion test (LIT) bioassay from engorged females collected from GI. The area under the concentration vs. time curves (AUC_0–35d) obtained post-second treatment was 1.51 ± 0.39-fold higher than those observed post-first treatment (P<0.05). The mean plasma concentrations of ivermectin 3.15% at 20 days after the first, second and third treatment were 17.0, 27.5 and 37.8 ng/mL, respectively (P<0.01). The elimination half-life of ivermectin post-third treatment was significantly longer than that was previously reported after a single dose (P<0.01). Values of therapeutic efficacy percentage reached 75.6% post-first treatment and between 95.9 and 100% after the second treatment. Ticks evaluated by LIT showed a significant increase in lethal concentrations after treatments. Although the efficacy level was high, the successive treatments with long-acting ivermectin formulation generate a significant accumulation of drug in plasma and could increase the levels of resistance to this drug in the tick population.     

Combination of cypermethrin and thymol for control of Rhipicephalus microplus: Efficacy evaluation and description of an action mechanism

The cattle tick Rhipicephalus microplus is one of the most important ectoparasites in the tropical and subtropical regions of the world. Synthetic pyrethroids are widely used to control this tick, and the selection of resistant populations is a huge problem worldwide. The activity of thymol, a natural monoterpene, free or in combination with other compounds, has been demonstrated against different species of ticks. However, the mode of action is not fully understood. The present study aimed to evaluate the efficacy and the potential mode of action of the combination of cypermethrin and thymol on ticks from two populations with different levels of susceptibility to cypermethrin (low and high susceptibility). The isolated acaricidal activity of cypermethrin and thymol on larvae was carried out in different concentrations. The combination with different concentrations of cypermethrin and fixed concentrations of thymol (1300 µg/mL for the low susceptibility population; 690 µg/mL for the high susceptibility population) were performed. Adult engorged females were divided into five experimental groups (n = 20): 1) Control group untreated; 2) Control group: 2.0% (v/v) DMSO; 3) Thymol group: 1300 µg/mL thymol; 4) Cypermethrin group: 3700 µg/mL cypermethrin; 5) Association of cypermethrin (3700 µg/mL) + thymol (1300 µg/mL). A subgroup was used to study the efficacy of the reproductive parameters and another subgroup, with ten adults from each treatment, was used to quantify thymol and cypermethrin by HPLC chromatographic analysis. All compounds tested were effective on larvae from both populations, and the combination with thymol decreased the LC₅₀ of cypermethrin (232.4 to 52.7 µg/mL) on the low-susceptibility population. The combination of thymol and cypermethrin was effective in both populations of R. microplus (reproductive performance of engorged females) when compared to the untreated control group, even with higher percent control values (pop. 1: 93.5 ± 5.6% and pop. 2: 92.7 ± 1.1%) than the group treated only with cypermethrin (pop. 1: 87.3 ± 7.3% and pop. 2: 83.5 ± 1.2%). From the HPLC analyzes, a higher concentration of cypermethrin (pop. 1: 30.3 ± 6.9 and pop. 2: 45.4 ± 17.7 ng/mg) was detected in the tissues of engorged females treated with the combination compared to analyte concentrations in groups treated with cypermethrin only (pop. 1: 12.4 ± 4.4 pop. 2: 25.5 ± 9.4 ng/mg). This was the first study to investigate the acaricidal efficacy of the combination of thymol + cypermethrin on R. microplus and demonstrate that the presence of thymol increases the concentration of cypermethrin in the internal tissues of engorged females through a possible mechanism for increasing the penetration of cypermethrin at the cuticular level.     

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.     

Differential recognition by tick-resistant cattle of the recombinantly expressed Rhipicephalus microplus serine protease inhibitor-3 (RMS-3)

Rhipicephalus microplus is an important bovine ectoparasite, widely distributed in tropical and subtropical regions of the world causing large economic losses to the cattle industry. Its success as an ectoparasite is associated with its capacity to disarm the antihemostatic and anti-inflammatory reactions of the host. Serpins are protease inhibitors with an important role in the modulation of host–parasite interactions. The cDNA that encodes for a R. microplus serpin was isolated by RACE and subsequently cloned into the pPICZαA vector. Sequence analysis of the cDNA and predicted amino acid showed that this cDNA has a conserved serpin domain. B- and T-cell epitopes were predicted using bioinformatics tools. The recombinant R. microplus serpin (rRMS-3) was secreted into the culture media of Pichia pastoris after methanol induction at 0.2 mg l^?1. qRT-PCR expression analysis of tissues and life cycle stages demonstrated that RMS-3 was mainly expressed in the salivary glands of female adult ticks. Immunological recognition of the rRMS-3 and predicted B-cell epitopes was tested using tick-resistant and susceptible cattle sera. Only sera from tick-resistant bovines recognized the B-cell epitope AHYNPPPPIEFT (Seq7). The recombinant RMS-3 was expressed in P. pastoris, and ELISA screening also showed higher recognition by tick-resistant bovine sera. The results obtained suggest that RMS-3 is highly and specifically secreted into the bite site of R. microplus feeding on tick-resistant bovines. Capillary feeding of semi-engorged ticks with anti-AHYNPPPPIEFT sheep sera led to an 81.16% reduction in the reproduction capacity of R. microplus. Therefore, it is possible to conclude that R. microplus serpin (RMS-3) has an important role in the host–parasite interaction to overcome the immune responses in resistant cattle.     

Cloning and characterization of Rhipicephalus appendiculatus voraxinα and its effect as anti-tick vaccine

Male tick-derived voraxinα and voraxinβ, a pair of testicular proteins, are transferred to female via copulation to stimulate female blood feeding in the tick Amblyomma hebraeum (A. hebraeum). Immunized animals with recombinant (r-)voraxinα and voraxinβ have been shown as highly resistant to the tick infestation. In this study, we describe the cloning and characterization of voraxinα homologue from the tick Rhipicephalus appendiculatus (R. appendiculatus), the major vector for East Coast fever in Eastern Africa. The sequence analysis of the R. appendiculatus voraxinα indicated that the deduced amino acid sequence had high similarity with voraxinα of the tick A. hebraeum and Dermacentor variabilis, suggesting that voraxinα is conserved in different tick genera. Quantitative RT-PCR and Western blotting analysis showed that male voraxinα was predominantly expressed in testis and its expression was induced by blood feeding. R. appendiculatus voraxinα was not secreted into the host during tick feeding and was detected in mated female hemolymph as measured by Western blotting. Preliminary vaccination of rabbits with r-voraxinα elicited the humoral immunity and conferred protective immunity against female ticks, resulting in the reduced fed weight. These results suggest that r-voraxinα could be a good candidate as anti-tick vaccine.     

Identification and functional analysis of ferritin 2 from the Taiga tick Ixodes persulcatus Schulze

Ferritin 2 (FER2) is an iron storage protein, which has been shown to be critical for iron homeostasis during blood feeding and reproduction in ticks and is therefore suitable as a component for anti-tick vaccines. In this study, we identified the FER2 of Ixodes persulcatus, a major vector for zoonotic diseases such as Lyme borreliosis and tick-borne relapsing fever in Japan, and investigated its functions. Ixodes persulcatus-derived ferritin 2 (Ip-FER2) showed concentration-dependent iron-binding ability and high amino acid conservation, consistent with FER2s of other tick species. Vaccines containing the recombinant Ip-FER2 elicited a significant reduction of the engorgement weight of adult I. persulcatus. Interestingly, the reduction of engorgement weight was also observed in Ixodes ovatus, a sympatric species of I. persulcatus. In silico analyses of FER2 sequences of I. persulcatus and other ticks showed a greater similarity with I. scapularis and I. ricinus and lesser similarity with Hyalomma anatolicum, Haemaphysalis longicornis, Rhipicephalus microplus, and R. appendiculatus. Moreover, it was observed that the tick FER2 sequences possess conserved regions within the primary structures, and in silico epitope mapping analysis revealed that antigenic regions were also conserved, particularly among Ixodes spp ticks. In conclusion, the data support further protective tick vaccination applications using the Ip-FER2 antigens identified herein.     

Chitosan-coated poly (Ɛ-caprolactone) nanoparticles as acaricide carriers

Among many species of ticks that affect beef and dairy cattle, Rhipicephalus (Boophilus) microplus is the most common. It is responsible for heavy losses in milk and meat production. In this work we introduce nanostructures such as chitosan-poly-?-caprolactone (CS_PCL) nanoparticles to encapsulate amitraz (CS_PCLnp_Amitraz) and fluazuron (CS_PCLnp_Fluazuron) to treat tick infestations more effectively. The CS_PCLnp_Amitraz system has a final amitraz concentration of 1.0 mg/mL with a particle size of 275 ± 30 nm, surface charge of +43 ± 7 mV and entrapment efficiency of 77 ± 1%. The CS_PCLnp_Fluazuron system has a drug concentration of 0.5 mg/mL with a particle size of 295 ± 35 nm, surface charge of +45 ± 10 mV and entrapment efficiency of 89 ± 1%. Both systems reduced cytotoxicity on Balb/c 3T3 culture cells and were also active against R. microplus. Both molecules - amitraz and fluazuron – formed molecularly dispersed active compounds inside the core of the PCL polymer matrix. The PCL surface was composed of a chitosan layer, which influenced the stability of the steric nanoparticles at pH greater than 7. Both systems were stable at a saline concentration of 1.25 mol/L and at temperatures below 50 °C. Experiments conducted in vivo with CS_PCLnp_Amitraz, at doses of active ingredient equivalent to those of commercial products, showed decreased tick infestation for 21 days, as well as higher acaricide effect than observed for commercial products, which recommend a reapplication in 14 days. The acaricide effect was even stronger when CS_PCLnp_Amitraz (same dose as for commercial products) and CS_PCLnp_Fluazuron (half of the amount for commercial products) were administered together.     

Rhipicephalus microplus cystatin as a potential cross-protective tick vaccine against Rhipicephalus appendiculatus

Rhipicephalus appendiculatus, the brown ear tick, is an important disease vector of livestock in eastern, central and southern Africa. Rhipicephalus appendiculatus acaricide resistance requires the search for alternative methods for its control. Cystatins constitute a superfamily of cysteine peptidase inhibitors vital for tick blood feeding and development. These inhibitors were proposed as antigens in anti-tick vaccines. In this work, we applied structural and biochemical approaches to characterize a new cystatin named R. appendiculatus cystatin 2a (Racys2a). Structural modeling showed that this new protein possesses characteristic type 2 cystatin motifs, besides conservation of other structural patterns along the protein. Peptidase inhibitory assays with recombinant Racys2a showed modulation of tick and host cathepsins involved in blood digestion and immune system responses, respectively. A heterologous tick challenge with R. appendiculatus in rabbits immunized with recombinant Rhipicephalus microplus cystatin 2c (rBmcys2c) was performed to determine cross-reactivity. Histological staining showed that rBmcys2c vaccination caused damage to the gut, salivary gland and ovary tissues in R. appendiculatus. Furthermore, cystatin vaccine reduced the number of fully engorged adult females in 11.5 %. Consequently, strategies to increase the protection rate are necessary, including the selection of two or more antigens to compose a vaccine cocktail.     

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.     

Functional characterization of candidate antigens of Hyalomma anatolicum and evaluation of its cross-protective efficacy against Rhipicephalus microplus

Hyalomma anatolicum and Rhipicephalus microplus seriously affect dairy animals and immunization of host is considered as a sustainable option for the management of the tick species. Identification and validation of protective molecules are the major challenges in developing a cross-protective vaccine. The subolesin (SUB), calreticulin (CRT) and cathepsin L-like cysteine proteinase (CathL) genes of H. anatolicum were cloned, sequenced and analysed for sequence homology. Both Ha-SUB and Ha-CRT genes showed very high level of homogeneity within the species (97.6–99.4% and 98.2–99.7%) and among the tick species (77.3–99.3% and 85.1–99.7%) while for Ha-CathL the homogeneity was lower among ticks (57.5–89.5%). Besides tick species, both Ha-SUB and Ha- CRT genes showed high level of homogeneity with dipterans (47.2–53.4% and 72.0–74.4%) and nematodes (64.0% by CRT). The level of expression of the conserved genes in different stages of the tick species was studied. The differences in fold change of expression (FCE) of the targeted genes in life stages of tick were not statistically significant except Ha-SUB in eggs and in frustrated females, Ha-CRT in fed male and Ha-CathL in unfed and frustrated females where highest FCE was recorded. The functional properties of the genes were studied by RNAi technology and a significant level of gene suppression (p < 0.05) resulted in very low percentage of engorgement of treated ticks viz., 3.7%, 11.1% and 30.0% in Ha-SUB, Ha-CRT and Ha-CathL respectively, in comparison to control was recorded. The recombinant proteins rHa-SUB, rHa-CRT and rHa-CathL encoded by the genes were expressed in prokaryotic expression system. They were evaluated for cross-protective efficacy and found to be respectively, 65.4%, 41.3% and 30.2% protective against H. anatolicum and 54.0%, 37.6% and 22.2%, against R. microplus infestations.     

Cercopithifilaria spp. in ticks of companion animals from Asia: new putative hosts and vectors

Cercopithifilaria bainae, Cercopithifilaria grassi, and Cercopithifilaria sp. II sensu Otranto et al., 2013 tick borne filarioids are typically found in dogs. Among them, Cercopithifilaria bainae has a worldwide distribution according to the occurrence of its tick vector, Rhipicephalus sanguineus sensu lato (s.l.). Nevertheless, in Asian countries, despite the wide presence of this tick species, data on Cercopithifilaria spp. are scant. Therefore, this study aimed to assess the occurrence of these dermal filarioids in ixodid ticks collected on dogs and cats from Asian countries, providing a better epidemiological picture on their distribution in this continent. Ticks (n = 687) of the species Rhipicephalus sanguineus s. l. (n = 667), Rhipicephalus haemaphysaloides (n = 8), Haemaphysalis longicornis (n = 7), Haemaphysalis campanulata (n = 1), Haemaphysalis wellingtoni (n = 2), Haemaphysalis hystricis (n = 1), and Ixodes sp. (n = 1) were collected on dogs and cats under the frame of previous studies in China, India, Indonesia, Malaysia, Singapore, Taiwan, Thailand, the Philippines and Vietnam. Tick samples were molecularly screened for Cercopithifilaria spp. by conventional PCR and real-time PCR using two pair of primers targeting partial sequences of cytochrome c oxidase 1 gene. Overall, Cercophitifilaria spp. DNA was detected in 9.5% (n = 65/687) of the tick specimens tested, with C. bainae being the most prevalent species (8.9%), followed by C. grassii (0.6%). Most Cercophitifilaria spp. positive ticks were collected on dogs (92.3%; 60/65); whereas ticks collected on cats represented 7.7% of the positive specimens. In addition, Cercopithifilaria spp. were mostly detected in R. sanguineus s.l. ticks (96.9%; 63/65), followed by Rhipicephalus haemaphysaloides (3.1%; 2/65). Data herein presented demonstrate the occurrence of dermal tick borne filarioids of the genus Cercopithifilaria in several Asian countries, with C. bainae being the most prevalent species. We also report for the first time the molecular detection of C. bainae in R. sanguineus s.l. ticks collected on cats, as well as in R. haemaphysaloides ticks, suggesting that the biological cycle of this filarioid species may involve other intermediate and definitive hosts than R. sanguineus s.l. and dogs. However, confirmatory studies on the role of other tick species and domestic cats on the biology of C. bainae are advocated.