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On-line version ISSN 1678-8060
Mem. Inst. Oswaldo Cruz vol.104 no.7 Rio de Janeiro Nov. 2009
Carlos AN RamosI, II; Flábio R AraújoII, +; Ingrid IF SouzaII; Renato HM OliveiraII; Carina EliseiII; Cleber O SoaresII; Ana MS SaccoIII; Grácia MS RosinhaII; Leucio C AlvesI
IDepartamento de Medicina Veterinária, Universidade Federal Rural de Pernambuco, Recife, PE, Brasil
IIEmbrapa Gado de Corte, BR262 km4 CP154, 79002970 Campo Grande, MS, Brasil
IIIEmbrapa Pecuária Sul, Bagé, RS, Brasil
Babesia bovis is a tick-borne pathogen that remains an important constraint for the development of cattle industries in tropical and subtropical regions of the world. Effective control can be achieved by vaccination with live attenuated phenotypes of the parasite. However, these phenotypes have a number of drawbacks, which justifies the search for new, more efficient immunogens based mainly on recombinant protein technology. In the present paper, ribosomal phosphoprotein P0 from a Brazilian isolate of B. bovis was produced and evaluated with regard to conservation and antigenicity. The protein sequence displayed high conservation between different Brazilian isolates of B. bovis and several Apicomplexa parasites such as Theileria, Neospora and Toxoplasma. IgG from cattle experimentally and naturally infected with B. bovisas well as IgG1 and IgG2 from naturally infected cattle reacted with the recombinant protein. IgG from cattle experimentally infected with Babesia bigemina cross-reacted with B. bovis recombinant P0. These characteristics suggest that P0 is a potential antigen for recombinant vaccine preparations against bovine babesiosis.
Key words: Babesia bovis - babesiosis - bovine - recombinant ribosomal phosphoprotein
Bovine babesiosis caused by Babesia bovis and Babesia bigemina continues to be an economically important disease with wide distribution in tropical and subtropical regions, where the common arthropod vector Rhipicephalus microplus (Murrel & Barker 2003) occurs (Bock et al. 2004). B. bovis causes the most pathogenic disease among parasites of the genus, resulting from sequestration of infected erythrocytes in the microcapillary endothelia of vital organs and a hypotensive shock syndrome denominated cerebral babesiosis, which is often fatal to susceptible cattle (Wright & Goodger 1988, Clark & Jacobsen 1998). Cattle that survive initial infection, either naturally or following chemotherapy, remain persistently infected and resistant to clinical disease, thereby allowing the use of attenuated phenotypes as live vaccines. However, these vaccines have limitations, including the transmission of other blood-borne pathogens and failure due to vaccine breakdowns (de Vos & Bock 2000), which justifies the search for new, more efficient immunogens.
Advances in molecular techniques have led to the discovery and evaluation of many subunit antigens of B. bovis in recent years, which have provided antigens of the parasite with immunogenic and antigenic potential (Gaffar et al. 2004, Brown et al. 2006, Brayton et al. 2007, Bono et al. 2008). P0 is a neutral protein found in all eukaryotic organisms (Rich & Steitz 1987) and is a component of a family of ribosomal phosphoproteins, together with P1 and P2 (Rajeshwari et al. 2004). P0 (but not P1 and P2) is of vital importance to cells, as demonstrated in Saccharomyces cerevisiae (Santos & Ballesta 1994). Although a ribosomal component, this protein has been located on the surface of many eukaryotic cells, including many protozoan parasites (Singh et al. 2002), which may at least partially explain the antigenicity and immunogenicity of P0 against Plasmodium yoellii, Leishmania major and Babesia microti (Chatterjee et al. 2000, Iborra et al. 2005, Terkawi et al. 2007b). Moreover, in vitro studies have demonstrated that anti-P0 antiserum can neutralise Toxoplasma gondii, Neospora caninum and B. bovis parasites by either inhibiting their growth or blocking cell invasion (Sehgal et al. 2003, Terkawi et al. 2007a, Zhang et al. 2007). Thus, we hypothesise that P0 is a potential candidate as an immunogen against bovine babesiosis.
The aims of the present paper were to analyse the genetic conservation of the P0 gene among Brazilian isolates of B. bovis and produce recombinant P0 (rPO) from a Brazilian isolate and evaluate its antigenicity with sera from cattle naturally or experimentally infected with B. bovis and sera from cattle experimentally infected with B. bigemina in an enzyme-linked immunosorbent assay (ELISA).
MATERIAL AND METHODS
Isolates - Five Brazilian isolates of B. bovis were used. The isolates were from the states of Bahia, São Paulo, Rio Grande do Sul (RS), Mato Grosso do Sul and Rondônia, representing all physiographic Regions of Brazil (Northeast, Southeast, South, Midwest and North, respectively).
Gene sequencing and analysis - Whole ORFs of the P0 gene from the five Brazilian isolates of B. bovis were amplified by polymerase chain reaction (PCR) and cloned into pGEM-T Easy (Promega, USA) for sequence analysis, following the manufacturer's instructions. Amplifications of the whole gene were performed in two steps using the primer sets P0-1F 5'-ATGGCTCGCATGAGCAAG-3' and P0-1R 5'-ACCCTGTCGTTAACCTTGATGA-3' and P0-2F 5'-ATGTCCACCTCATCAAGGTTAAC-3' and P0-2R 5'-TTAGTCAAAGAGGGAGAATCCC-3' to produce two amplicons. Amplification reactions were performed in a volume of 25 μL containing 10 mM Tris-HCl (pH 8.3), 50 mM KCl, 1.5 mM MgCl2, 0.2 mM of each deoxynucleoside triphosphate, 12 pmol of each primer, 100 ng of genomic DNA and 1.25 U of Taq DNA polymerase (Cenbiot, Brazil). Amplifications were performed in a Mastercycler thermocycler (Eppendorf, Germany) as follows: 94°C for 1 min (denaturation); 30 cycles of 94°C for 1 min (denaturation), 53ºC for 30 sec (annealing) and 72°C for 40 sec (extension) and a final cycle at 72°C for 4 min (extension). PCR products were analysed by electrophoresis on 1% agarose gels stained with SybrGold (Invitrogen, USA).
The DNA sequences of P0 from five Brazilian isolates of B. bovis were obtained using the BigDye Terminator kit (Applied Biosystems, USA). Sequences were assembled using the Sequencher v.4.1.4 software program (Gene Codes, USA) and submitted to BLASTn search (http://www.ncbi.nlm.nih.gov) to verify the sequence identity. Multiple sequence alignment was performed with the ClustalW algorithm (www.ebi.ac.uk/Tools/clustalw2/index.html).
Phylogenetic analyses were conducted with deduced amino acid sequences of P0 from Brazilian isolates of B. bovis, other species of Babesia, other Apicomplexa and Bos taurus using the MEGA software program, version 4.0 (Tamura et al. 2007). A phylogenetic tree was generated using the neighbour-joining method (Saitou & Nei 1987) and a bootstrap resampling technique of 1,000 replications was performed to statistically support the reliabilities of the nodes on the trees (Felsenstein 1985).
Cloning and expression - DNA from the southern Brazilian isolate of B. bovis was used to amplify the complete ORF of P0 by PCR using the primer set P0-1F and P0-2R. The amplification reaction was performed in a volume of 25 μL, as described above, with an initial denaturing step of 95°C for 3 min followed by 35 cycles of 95°C for 1 min, 58°C for 30 sec and 72°C for 1 min and a final extension step at 72°C for 4 min.
The amplicon was initially cloned into pGEM-T Easy (Promega, USA) and subsequently sub-cloned into the pRSET-c (Invitrogen, USA) EcoRI site. Chemically competent Escherichia coli host strain BL-21 cells were transformed with recombinant plasmid (pRSET-c/P0) and the transformation was spread on selective plates containing Luria Bertani (LB) medium supplemented with 100 μg/mL ampicillin and 34 μg/mL chloramphenicol. The expression of the P0 gene was carried out in LB broth with the addition of 1 mM isopropyl-β-D-galactopyranoside.
The purification of rP0 was carried out with agarose-Ni+ resin (ProBond, Invitrogen, USA), following the manufacturer's instructions. The purified rP0 was analysed by sodium dodecyl-polyacrylamide gel electrophoresis (SDS-PAGE) and Western blotting with monoclonal anti-histidine antibody (GE, UK).
Protein concentration was determined by SDS-PAGE stained with Coomassie blue by comparison with known concentrations of bovine serum albumin using the LabImage v.3.3.2 image analysis software (Loccus, Brazil).
Serum samples - Cattle serum samples negative for antibodies to B. bovis and B. bigemina were collected from tick-free areas of RS (n = 33) and Paraíba (n = 29), Brazil and from cattle kept in a tick-free isolation area of Embrapa Beef Cattle, Campo Grande, MS, Brazil (n = 67). Serum samples from cattle naturally infected with B. bovis were obtained from state of Pará (PA), Brazil (n = 133). These serum samples were analysed by a crude B. bovis antigen ELISA (Madruga et al. 2000). Serum samples from cattle experimentally infected with B. bovis (n = 77) or B. bigemina (n = 36) were obtained from the South Embrapa Cattle and Sheep Research Centre, Bagé, RS, Brazil. These animals were raised in a tick-free area and were periodically tested for antibodies to B. bovis, B. bigemina and Anaplasma marginale by the immunofluorescent antibody test and examination of stained blood smears. Experimental infections with B. bovis or B. bigemina were carried out by the subcutaneous route with pure isolates kept in liquid nitrogen and sera were collected 30 days post-inoculation.
Standardisation of ELISA with rP0 - Optimal dilutions of antigen, sera and conjugate were determined using six sera negative and six sera positive for antibodies against B. bovis. Then, 96-well Costar 3590 plates (Corning, USA) were adsorbed with 1 ng/mL of rP0 (100 μL/well) diluted in phosphate buffer containing 0.1% Tween 20 (PBST) for 12 h at 4°C. Plates were blocked with PBST with 5% skim milk for 1 h at 37°C. After five washes with PBST, control and test sera (in duplicate) - diluted 1:800 in PBST - were incubated for 60 min at 37°C. Plates were then washed five times with PBST and 100 μL/well of rabbit anti-bovine IgG horseradish peroxidase conjugate (Sigma A5295, USA) diluted 1:10,000 in PBST was added. Plates were incubated for 30 min at 37°C and, after five washes with PBST, reactions were revealed with Fast-OPD (50 μL/well; Sigma, USA). The reaction was stopped by the addition of 100 μL/well of H2SO4 (2.5 N) and results were read on an EL-800 ELISA reader (Bio-Tek, USA) with a 490-nm filter.
IgG1 and IgG2 titration - For titration of anti-rP0 IgG1 and IgG2, sera from 30 cattle naturally infected with B. bovis from PA were tested as described above, except that sera were diluted from 1:100-1:3,800, anti-bovine IgG1 and IgG-2 monoclonal antibodies (Serotec, USA) were diluted 1:5,000 and rabbit anti-mouse IgG horseradish conjugate (Sigma A-9044, USA) was diluted 1:10,000. Titres were defined as the reciprocal of the highest dilution giving a positive result.
Cutoff selection - The cutoffs of the ELISA tests were determined based on those described by Frey et al. (1998).
Statistical analysis - The means of the optical densities of sera tested for IgG were analysed by ANOVA followed by Tukey's test, with a 95% confidence level. Titres of IgG1 and IgG2 were analysed by Wilcoxon signed-rank test, with a 95% confidence level.
RESULTS AND DISCUSSION
The P0 gene sequences (939 nucleotides) obtained in the present study were deposited in the Genbank database under the following accession: FJ588004 Midwestern isolate, FJ588005 Northeastern isolate, FJ588006 Northern isolate, FJ588007 Southeastern isolate and FJ588008 Southern isolate of B. bovis.
The multiple alignment of P0 DNA sequences revealed a high degree of identity among the Brazilian isolates of B. bovis (> 99%). The best hit by BLASTn was the Genbank entry AF498365 (99% nucleotide identity). The amino acid sequence homology among Brazilian isolates was 100%. This demonstrates that P0 is highly conserved among isolates of B. bovis, which is a key pre-requisite for any subunit vaccine candidate.
The P0-deduced amino acid sequences of the Brazilian isolates of B. bovis were aligned with P0-deduced amino acid sequences of B. bovis USA AAM18123, Babesia caballi BAF91359, B. bigemina BAF91357, Babesia gibsoni BAF45423, Babesia equi BAF91358, Theileria annulata XP_954430, N. caninum BAF62528, T. gondii AAK69358 and B. taurus AAI51696. A considerable level of homology was observed between the P0 sequences of the Brazilian isolates of B. bovis and B. caballi (85%), B. bigemina (84%), B. gibsoni (80%), B. equi (67%), T. annulata (62%), N. caninum (58%) and T. gondii (58%). This high degree of homology may explain the serological cross-reactivity or cross-protective immunity between the P0 proteins of B. gibsoni and B. microti (Terkawi et al. 2007b), B. gibsoni and B. rodhaini (Terkawi et al. 2008), B. gibsoni and B. bovis (Terkawi et al. 2007a) and others. The high degree of homology also suggests that P0 may be a universal immunogen against bovine babesiosis caused by B. bovis and B. bigemina. Nevertheless, there was considerable homology between the P0 proteins from B. bovis and B. taurus (46%). This indicates the possibility that immunisation with rP0 from B. bovis may generate autoimmune antibodies in cattle. The phylogenetic analysis resulted in a tree with two branches (Fig. 1). The first one included Apicomplexa parasites, with a subgroup containing all the isolates of B. bovis (Brazilian and North American), another subgroup with B. bigemina, B. caballi and B. gibsoni, a third subgroup with T. annulata and B. equi and the last one with the Sarcocystidae N. caninum and T. gondii. The second branch was of B. taurus.
The high degree of P0 conservation among protozoa and even in mammals, suggests that this protein plays an important role in cell metabolism. P0 has been associated with DNA repair, cell development, apoptosis and carcinogenesis (Brockstedt et al. 1998, Frolov & Birchler 1998, Liliensiek et al. 1998). Moreover, P0 is a structural component, together with P1 and P2, of the 60S ribosomal subunit of eukaryotes (Uchiumi et al. 1987) and, in knockout studies on S. cerevisiae, P0 was shown to be vital to cell survival (Santos & Ballesta 1994).
rP0 from B. bovis was produced as a 6x-histidine-tag fusion, resulting in a protein of approximately 37 kDa viewed by SDS-PAGE and western blotting (Fig. 2). This molecular mass is consistent with the fusion protein, including the his-tag (~3 kDa) and P0 (34 kDa). Using anti-sera against B. gibsoni P0, Terkawi et al. (2007a) detected protein bands of 31-34 kDa in lysates of B. gibsoni, B. bovis, B. bigemina, B. equi and B. caballi through western blot analysis.
The recognition of rP0 from B. bovis by cattle IgG was evaluated by ELISA (Fig. 3). Among the 77 sera from cattle experimentally infected with B. bovis, 75 (97.4%) were positive in the rP0 ELISA. Out of 133 sera from cattle naturally infected with B. bovis, 127 (95.5%) were positive. Out of 129 sera from cattle negative for B. bovis by ELISA with crude B. bovis antigen, 128 (99.2%) were also negative in the rP0 ELISA.
To investigate whether rP0 from B. bovis is recognised by B. bigemina antibodies, sera from cattle exclusively infected with B. bigemina were tested with rP0 by ELISA (Fig. 3). Out of 36 sera analysed, 34 (94.4%) were positive in the rP0 ELISA. Serological cross-reactions against P0 have been described among Babesia species (Terkawi et al. 2007a) and between Plasmodium falciparum and T. gondii (Sehgal et al. 2003). The cross-reaction found in the present paper suggests that P0 proteins from B. bovis and B. bigemina share immunodominant epitopes and raises the possibility that such epitopes may be involved in a cross-protective immune response.
rP0 from B. bovis was also recognised by IgG1 and IgG2 antibodies from 30 cattle naturally infected with B. bovis from PA, with a significantly higher titre of IgG2 (p = 0.0121) (Fig. 4). Th1 responses, including the production of interferon-γ, activation of macrophages and production of the IgG2 isotype by B cells, have been associated with immunity against intra-erythrocytic pathogens (Brown et al. 1998, Aguilar-Delfin et al. 2003).
There is the possibility that the IgG, IgG1 and IgG2 responses found in the present study may be elicited by cross-reactions with antibodies against P0 proteins from other Apicomplexa parasites, such as N. caninum. Nevertheless, only 0.8% of the sera collected from cattle raised in tick-free areas did not react with rP0, suggesting that cross-reactions are relevant only at the Babesia genus level.
In summary, the results of the present paper indicate the high conservation of B. bovis P0 not only between different B. bovis Brazilian isolates, but also between many protozoan parasites, as well as the antigenicity of rP0 with B. bovis and B. bigemina-infected sera. These two characteristics are desirable for the subunit vaccine.
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Received 26 May 2009
Accepted 23 September 2009
Financial support: EC (INCO 003691-MEDLABAB), CNPq, FUNDECT, FACEPE