Vaccination with an in vitro culture attenuated Babesia bovis strain safely protects highly susceptible adult cattle against acute bovine babesiosis

Abstract

IntroductionLive in vivo attenuated Babesia bovis vaccines produced by sequential passages in splenectomized calves have historically been used to control acute bovine babesiosis in endemic areas worldwide. However, several constraints prevent the widespread use of these vaccines, including the need for several splenectomized calves to produce vaccine batches, and potential inconsistent parasite attenuation, which contraindicates their use for highly Babesia-susceptible adult cattle. Thus, the use of vaccines based on well-defined in vitro culture attenuated B. bovis strains emerges as a more sustainable and efficient alternative. Previous work demonstrated that the culture attenuated strain Att-S74-T3Bo is non-tick transmissible and able to safely protect calves against needle challenge with a B. bovis virulent strain.Methods and resultsHerein we evaluated safety and efficacy of Att-S74-T3Bo in preventing acute babesiosis in adult (>1.5 year of age) cattle. Results demonstrated that Att-S74-T3Bo vaccination of adult animals (n=5) induced self-limiting signs of acute infection and protected the vaccinated animals against challenge with the homologous virulent B. bovis strain Vir-S74-T3Bo. Att-S74-T3Bo-vaccinated adult cattle developed significant (P<0.05) monocytosis, with concomitant neutropenia and CD4+ leukopenia, in peripheral blood early after vaccination. Also, vaccinated animals developed a specific signature of pro- and anti-inflammatory cytokine expression in peripheral blood and significant levels of IgM, total IgG, IgG1, and IgG2 against the B. bovis immunodominant antigen RAP-1 CT. Strikingly, none of the vaccinated animals showed any signs of acute babesiosis after challenge with Vir-S74-T3Bo. In contrast, control adult cattle (n=5) showed pathognomonic symptoms of acute babesiosis, and significant decrease (P<0.05) in lymphocytes, monocytes, and neutrophils, starting on day 7 post-challenge. All control animals developed severe acute disease and were euthanized on days 10 through 12 days post-challenge.Discussion and conclusionEvidence from this study indicates that Att-S74-T3Bo safely protects highly susceptible adult cattle against challenge with a homologous virulent strain of B. bovis. In conclusion, Att-S74-T3Bo may be considered as a potential efficient and sustainable attenuated candidate vaccine strain to control acute bovine babesiosis in highly susceptible adult cattle. Future studies should focus on increasing the number of animals vaccinated, duration of immunity, and efficacy of this attenuated strain against heterologous virulent parasite strains.

A conserved motif in the immune-subdominant RAP-1 related antigen of Babesia bovis contains a B-cell epitope recognized by antibodies from protected cattle

“…We then examined whether B-cell epitopes in the conserved 15-mer region of RRA are also recognized by antibodies in cattle protected from B. bovis infection. To this end, we analyzed IgM ad IgG responses in sera derived from calves and adult cattle that were vaccinated with in vitro culture attenuated parasites, and then challenged with a virulent strain of B. bovis in previous studies ( 30 , 31 ). We compared antibody responses against a synthetic peptide representing the sequence of the 15-mer motif of the B. bovis RRA, and a peptide representing an immunodominant B-cell epitope expressed in the CT end of RAP-1, recognized by monoclonal antibody BABB75 ( 12 ).…”

“…We compared antibody responses against a synthetic peptide representing the sequence of the 15-mer motif of the B. bovis RRA, and a peptide representing an immunodominant B-cell epitope expressed in the CT end of RAP-1, recognized by monoclonal antibody BABB75 ( 12 ). Antibodies in the calf sera were examined at different dates following immunization with an attenuated live vaccine and challenge with virulent B. bovis parasites ( 30 , 31 ).…”

“…Pre-immune and immune sera from the B. bovis experimentally infected cattle used in the ELISA analysis were generated in a previous study ( 30 , 31 ).…”

“…After addition of 55 μl TMB stop solution (SeraCare, Milford, MA), absorbance was measured at 450 nm using the SpectraMax ® 190 plate reader (Molecular Devices, San Jose, CA). The serum samples analyzed hereby were generated in a previous study ( 30 , 31 ) involving vaccination of cattle with the in vitro culture attenuated strain of B. bovis Att- S74-T3Bo B. bovis that was challenged with a virulent strain of the parasite Vir-S74-T3Bo B. bovis . Statistical significance was determined using a paired 2 tails type 1, T-test.…”

“…For permeabilized IFA, samples were first smeared on a slide, fixed for 5 min in 100% acetone, and then incubated with 0.1% Triton X-100. The slides were then incubated in 10% BSA for 1 h with a combination of anti-RAP-1 antibodies (2 ug/mL) and anti-RRA peptide antibodies (1/500) ( 31 ). The slides were washed three times with PBS and incubated in 10% BSA with goat anti-rabbit IgG Alexa Fluor ® 555 (red fluorescence) and goat anti-mouse IgG Alexa Fluor ® 488 (green fluorescence) (Thermo Fisher Scientific, Waltham, MA).…”