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.

Kumar B ，Manjunathachar HV ，Nagar G ，Ravikumar G ，de la Fuente J ，Saravanan BC ，Ghosh S ... -  《-》

Subolesin: a candidate vaccine antigen for the control of cattle tick infestations in Indian situation.

Identification of cross-protective tick vaccine antigens is a challenging area of veterinary research. To address this challenge, a recently identified candidate tick protective antigen, Subolesin (SUB), was targeted in this research. The conservation of subolesin ortholog of Hyalomma anatolicum and Rhipicephalus (Boophilus) microplus across different Indian strains was 98.1-99.4% (within species), while at the amino acid level SUB sequence homology was ≥53.2% (between tick species). Recombinant R. (B.) microplus SUB (rBmSu) was produced in Escherichia coli and characterized. Cross-bred cattle male calves (N=10) were immunized with three doses of 100 μg each of the rBmSu emulsified in 10% Montanide 888 at monthly intervals on days 0, 30 and 60. The control group was injected with PBS in 10% Montanide 888. For the first tick challenge, calves were infested with larvae of R. (B.) microplus generated from 100mg eggs 2 weeks after last immunization (day 75). The immunization resulted in 16.3%, 8.0%, 9.4%, and 26.1% reduction in female tick numbers (DT), weight (DW), oviposition (DO) and egg fertility (DF), respectively, when compared to controls. In the subsequent challenge on day 105, DT, DW, DO and DF were reduced by 9.0%, 4.1%, 8.6%, and 24.2%, respectively, when compared to controls. The vaccine efficacy (E) was equal to 44.0% and 37.2% after the first and second challenges, respectively. The results showed a positive correlation between antibody titers for both total IgG and IgG1 and E in the second but not in the first tick challenge. These results suggested the possibility of developing a SUB-based vaccine for control of cattle tick infestations under Indian conditions.

Shakya M ，Kumar B ，Nagar G ，de la Fuente J ，Ghosh S ... -  《-》

Efficacy of Rhipicephalus (Boophilus) microplus Bm86 against Hyalomma dromedarii and Amblyomma cajennense tick infestations in camels and cattle.

The recombinant Bm86-based tick vaccines have shown their efficacy for the control of cattle ticks, Rhipicephalus (Boophilus) microplus and R. annulatus infestations. However, cattle ticks often co-exist with multi-host ticks such as Hyalomma and Amblyomma species, thus requiring the control of multiple tick infestations for cattle and other hosts. Vaccination trials using a R. microplus recombinant Bm86-based vaccine were conducted in cattle and camels against Hyalomma dromedarii and in cattle against Amblyomma cajennense immature and adult ticks. The results showed an 89% reduction in the number of H. dromedarii nymphs engorging on vaccinated cattle, and a further 32% reduction in the weight of the surviving adult ticks. In vaccinated camels, a reduction of 27% and 31% of tick engorgement and egg mass weight, respectively was shown, while egg hatching was reduced by 39%. However, cattle vaccination with Bm86 did not have an effect on A. cajennense tick infestations. These results showed that Bm86 vaccines are effective against R. microplus and other tick species but improved vaccines containing new antigens are required to control multiple tick infestations.

Rodríguez-Valle M ，Taoufik A ，Valdés M ，Montero C ，Ibrahin H ，Hassan SM ，Jongejan F ，de la Fuente J ... -  《-》

Molecular characterization of Bm86 gene orthologs from Hyalomma excavatum, Hyalomma dromedarii and Hyalomma marginatum marginatum and comparison with a vaccine candidate from Hyalomma scupense.

The ixodid ticks from the Hyalomma genus are important pests of livestock, having major medical and veterinary significance in Northern Africa. Beside their direct pathogenic effects, these species are vectors of important diseases of livestock and in some instances of zoonoses. Anti-tick vaccines developed in Australia and Cuba based on the concealed antigen Bm86 have variable efficacy against H. anatolicum and H. dromedarii. This variation in vaccine efficacy could be explained by the variability in protein sequence between the recombinant Bm86 vaccine and Bm86 orthologs expressed in different Hyalomma species. Bm86 orthologs from three Hyalomma tick species were amplified in two overlapping fragments and sequenced. The rate of identity of the amino acid sequence of Hm86, He86 and Hdr86, the orthologs of Bm86, respectively, in H. marginatum marginatum, H. excavatum and H. dromedarii, with the Bm86 proteins from Rhipicephalus microplus (Australia, Argentina and Mozambique) ranged between 60 and 66%. The obtained amino-acid sequences of Hmm86, He86 and Hdr86 were compared with the Hd86-A1 sequence from H. scupense used as an experimental vaccine. The results showed an identity of 91, 88 and 87% for Hmm86, He86 and Hdr86, respectively. A specific program has been used to predict B cells epitopes sites. The comparison of antigenic sites between Hd86-A1 and Hm86/Hdr86/He86 revealed a diversity affecting 4, 8 and 12 antigenic peptides out of a total of 28 antigenic peptides, respectively. When the Bm86 orthologs amplification protocol adopted in this study was applied to H. excavatum, two alleles named He86p2a1 and He86p2a2 were detected in this species. This is the first time that two different alleles of Bm86 gene are recorded in the same tick specimen. He86p2a1 and He86p2a2 showed an amino acid identity of 92%. When He86p2a1 and He86p2a2 were compared to the corresponding sequence of Hd86-A1 protein, an identity of 86.4 and 91.0% was recorded, respectively. When compared to He86, Hdr86 and Hm86, Bm86 used in commercial and experimental vaccines, showed a greater extent of diversity than noted when the same Hyalomma orthologs were compared to Hd86-A1. Although significant, these variations were less extensive within the Hyalomma genus. Accordingly, thus suggesting that Hd86-A1 vaccine candidate might be more appropriate to target Hyalomma tick species than corresponding Bm86 commercial vaccines. However, vaccination trials with both antigens are required to validate this hypothesis.

Ben Said M ，Galai Y ，Mhadhbi M ，Jedidi M ，de la Fuente J ，Darghouth MA ... -  《-》

Oral Vaccination With a Formulation Combining Rhipicephalus microplus Subolesin With Heat Inactivated Mycobacterium bovis Reduces Tick Infestations in Cattle.

Contreras M ，Kasaija PD ，Merino O ，de la Cruz-Hernandez NI ，Gortazar C ，de la Fuente J ... -  《Frontiers in Cellular and Infection Microbiology》