A combination of antibodies against Bm86 and Subolesin inhibits engorgement of Rhipicephalus australis (formerly Rhipicephalus microplus) larvae in vitro

Abstract

Background: Rhipicephalus microplus is a hard tick species that has a high impact on cattle health and production in tropical and subtropical regions. Recently, ribosomal DNA and morphological analysis resulted in the reinstatement of R. australis as a separate species from R. microplus. Both feed on cattle and can transmit bovine pathogens such as Anaplasma and Babesia species. The current treatment with acaricides is becoming increasingly less effective due to the emergence of resistant tick strains. A promising alternative can be found in the form of anti-tick vaccines. The available commercial vaccines can be used to control tick infestation, but the lack of a knockdown effect (> 90% reduction in tick numbers as seen with effective acaricides) hampers its widespread use, hence higher efficacious vaccines are needed. Instead of searching for new protective antigens, we investigated the efficacy of vaccines that contain more than one (partially) protective antigen. For screening vaccine formulations, a previously developed in vitro feeding assay was used in which R. australis larvae are fed sera that were raised against the candidate vaccine antigens. In the present study, the efficacy of the Bm86 midgut antigen and the cytosolic Subolesin (SUB) antigen were evaluated in vitro.

Results: Antiserum against recombinant Bm86 (rBm86) partially inhibited larval engorgement, whereas antiserum against recombinant SUB (rSUB) did not have any effect on feeding of larvae. Importantly, when larvae were fed a combination of antiserum against rBm86 and rSUB, a synergistic effect on significantly reducing larval infestations was found. Immunohistochemical analysis revealed that the rBm86 antiserum reacted with gut epithelium of R. australis larvae, whereas the antiserum against rSUB stained salivary glands and rectal sac epithelium.

Conclusions: Combining anti-Bm86 and anti-subolesin antibodies synergistically reduced R. australis larval feeding in vitro. Rhipicephalus australis is a one host tick, meaning that the larvae develop to nymphs and subsequently adults on the same host. Hence, this protective effect could be even more pronounced when larvae are used for infestation of vaccinated cattle, as the antibodies could then affect all three developmental stages. This will be tested in future in vivo experiments.

Keywords: Artificial tick feeding; Bm86; In vitro screening; R. australis; R. microplus; Subolesin; Vaccine.

Fig. 1

Recombinant Bm86 and subolesin and determination of antigen specific antibody titers. a Coomassie staining (left) and Western blot using anti-HIS mouse IgG (right) of purified subolesin (SUB) using a 4–20% Bis-Tris gel. b Anti-SUB antibody titration by sandwich ELISA; recombinant SUB was captured with anti-HIS mouse IgG and pooled SUB vaccinated cow serum was diluted to calculate endpoint titers. End-point titer cut-off (Bmin*2) is indicated by dashed line. c Coomassie staining (left) and Western blot using Bm86 (P. pastoris-produced) specific rabbit IgG (right) of recombinant Bm86. d Anti-Bm86 antibody titration by sandwich ELISA adapted from Trentelman et al. [35]. Bm86 (baculovirus produced) was captured with Bm86 (P. pastoris-produced) specific rabbit IgG and pooled SUB vaccinated cow serum was diluted to calculate endpoint titers. End point titer cut-off (Bmin*2) is indicated by dashed line

Fig. 2

Localization of Bm86 in unfed R. australis larvae. Cross sections (20× magnification) of larvae stained with: a rabbit anti-Bm86 serum (1:400 diluted) and b naïve rabbit serum (1:400 diluted). Sections were pre-treated with proteinase K for 30 min before antibody incubation. Antibodies bound specifically to midgut epithelium (indicated with arrows). Abbreviations: Syn, synganglion; MG, midgut; RS, rectal sac; A, acinus

Fig. 3

Localization of SUB in unfed R. australis larvae with polyclonal anti-SUB rabbit serum. a A cross-section (20× magnification) of a larva stained with rabbit anti-SUB serum (1:400 diluted). The square indicates the area depicted in higher magnification (40×) in b. IgG showed binding in the acini of the salivary glands, throughout the cytoplasm and within granules (indicated with arrows). c A cross section of a larva (20× magnification) stained with naïve rabbit serum (1:400 diluted). The square indicates the area depicted in higher magnification (40×) in d. d Details of the salivary glands are depicted on the right side (40× magnification). In contrast to the polyclonal anti-SUB serum no IgG binding could be observed in the salivary glands after incubation with naïve rabbit serum. Abbreviations: Syn, synganglion; MG, midgut; RS, rectal sac; A, acinus

Fig. 4

The effect of monospecific antisera against tick antigens and a 1:1 combination of these antisera on R. australis larval feeding as compared to tick naïve bovine serum. Larvae of R. australis were fed in vitro on 600 µl antisera against Bm86 or SUB or on 300 µl anti-Bm86 bovine serum combined with 300 µl anti-SUB bovine serum. After 48 h, ticks were visually scored for feeding. Bars represent the inhibition of feeding as compared to the control group, expressed as a percentage. Error bars represent the standard deviation. *P < 0.05, n.s., not significant

Fig. 5

The effect of one-time diluted monospecific antisera against tick antigens and a 1:1 mixture of these antisera on R. australis larval feeding as compared to tick naïve bovine serum. Larvae of R. australis were fed in vitro on 300 µl antisera against Bm86 or SUB diluted with 300 µl naïve bovine serum. Again, 300 µl anti-Bm86 bovine serum was combined with 300 µl anti-SUB bovine serum to assess synergistic effects of both antigens. After 48 h, ticks were visually scored for feeding. Bars represent the inhibition of feeding as compared to the control group, expressed as a percentage. Error bars represent the standard deviation