The B. microplus (Susceptible, CENAPA, Mexico strain) and B. annulatus (Mercedes, Texas, USA strain) ticks were obtained from laboratory colonies maintained at the University of Tamaulipas, Mexico. Originally, these tick strains were collected from infested cattle in Tapalpa, Jalisco, Mexico and Mercedes County, Texas, USA for B. microplus and B. annulatus, respectively. Ticks were maintained during two years at the facilities of the Faculty of Veterinary Medicine, University of Tamaulipas, where several generations of tick larvae were fed on cows and collected until repletion to allow for oviposition and hatching in humidity chambers at 12 hr light: 12 hr dark photoperiod, 22–25°C and 95% relative humidity. Larvae were 15 days of age at the time of infestations.
The recombinant Ba86 (Israeli strain) and Bm86 (Mozambique strain) were secreted in P. pastoris and purified as reported previously . Protein adjuvation was made by mixing a solution of anhydromannitoletheroctodecenoate (Montanide ISA 50 V; Seppic, Paris, France) with the recombinant protein solution in batch-by-batch processes using a high-speed mixer Heidolph DIAX 900 (Heidolph Elektro, Kelheim, Germany) at 8,000 rpm and the vaccine was filled manually under sterile conditions in glass bottles of 20 ml (Wheaton, Millville, NJ, USA) at a concentration of 100 μg/2 ml dose. Quality controls were made by testing mechanical and thermal stability of vaccine emulsions as described by Canales et al. . The commercial Bm86 (Cuban Camcord strain) vaccine (Gavac, Revetmex, Mexico City, Mexico) also contains 100 μg/2 ml dose of P. pastoris-derived purified recombinant protein formulated as described above.
Cattle immunization with recombinant proteins and tick infestations
Five crossbred calves per group were each immunized with 3 doses (weeks 1, 3 and 7) containing 100 μg/dose of purified recombinant proteins formulated as described above. Negative controls were injected with adjuvant/saline alone. Cattle were injected intramuscularly with 2 ml/dose using a 5 ml syringe and an 18G needle. Twelve days after the last immunization, cattle in vaccinated and control groups were infested with 10,000 B. annulatus (Mercedes, Texas, USA strain) and B. microplus (Susceptible, Mexico strain) larvae/animal applied individually to each animal in separate cotton cells attached to the back of the animals. Cattle were cared for in accordance with standards specified in the Guide for Care and Use of Laboratory Animals.
Data collection and evaluation
Adult female ticks dropping from cattle were daily collected, counted and weighted. All the collected adult female ticks were assessed for oviposition and egg fertility . The personnel collecting the ticks were 'blinded' as to which group animals belonged. The efficacy of vaccine formulations was evaluated employing the following formulae .
Effect on the number of adult female ticks (DT) = 100 [l-(NTV/NTC)], where NTV is the number of adult female ticks in the vaccinated group and NTC is the number of adult female ticks in the control group.
Effect on tick weight (DW) = 100 [1-(WTV/WTC)], where WTV is the average adult female tick weight in the vaccinated group and WTC is the average adult female tick weight in the control group.
Effect on oviposition (DO) = 100 [1-(PATV/PATC)], where PATV is the average weight of the eggs per survived tick in the vaccinated group and PATC is the average weight of the eggs per survived tick in the control group.
Effect on egg fertility (DF) = 100 [1-(PPLOV/PPLOC)], where PPLOV is the average weight of the larvae per gram of eggs in the vaccinated group and PPLOC is the average weight of the larvae per gram of eggs in the control group.
Vaccine efficacy (E) was calculated as 100 [l-(CRT × CR0 × CRF)], where CRT = NTV/NTC, CR0 = PATV/PATC and CRF = PPLOV/PPLOC that represent the reduction in the number of adult female ticks, oviposition and egg fertility as compared to the control group, respectively.
A Student's t-test with unequal variance (P = 0.05) was used to compare the results of adult female tick number, tick weight, oviposition and egg fertility between vaccinated and control groups.
Determination of serum antibody levels by ELISA
Before each immunization and 12 (before tick infestation) and 37 days after the last immunization, blood samples were collected from each calf into sterile tubes and maintained at 4°C until arrival at the laboratory. Serum was then separated after centrifugation and stored at -20°C. Serum antibody titers were determined using an antigen-specific indirect ELISA. Purified recombinant Bm86 (Mozambique strain) and Ba86 (Israeli strain) antigens (0.1 μg/well) were used to coat ELISA plates overnight at 4°C. Sera were serially diluted to 1:10, 1:100 and 1:1000 in PBST (PBS/0.5% Tween 20, pH 7.2) and 10% fetal bovine serum (Sigma). The plates were incubated with the diluted sera for 1 hr at 37°C and then incubated with 1:10,000 rabbit anti-bovine IgG-HRP conjugates (Sigma) for 1 hr at 37°C. The color reaction was developed with 3,3',5,5'-tetramethylbenzidine (Sigma) and the OD450 nm was determined. After incubation the plates were washed with PBST. Antibody titers were considered positive when they yielded an OD450 nm value at least twice as high as the preimmune serum. Antibody titers in immunized cattle were expressed as the OD450 nm value for the highest serum dilution (1:1000) and compared between vaccinated and control cattle using an ANOVA test (P < 0.05).
The sequences of the Ba86 (Mercedes, Texas, USA; Genbank accession number FJ456927) and Bm86 (Susceptible, Mexico; FJ456928) strains were determined as described previously . The protein sequences were aligned with Ba86 (Israeli strain; ABY58969), Bm86 (Mozambique strain; ABY58968) and Bm86 (Cuban Camcord strain in Gavac; ) using the program AlignX (Vector NTI Suite V 8.0, InforMax, Invitrogen, Carlsbad, CA, USA) with an engine based on the Clustal W algorithm . Antigenic peptides (=7 residues) were predicted using the method of Kolaskar and Tongaonkar , with a reported accuracy of about 75% http://immunax.dfci.harvard.edu/Tools/antigenic.pl.