Comparative Transcriptome Analysis of Babesia bigemina Attenuated Vaccine and Virulent Strains of Mexican Origin.

Bovine babesiosis, caused by the protozoan Babesia bigemina, is one of the most important hemoparasite diseases of cattle in Mexico and the world. An attenuated B. bigemina strain maintained under in vitro culture conditions has been used as a live attenuated vaccine; however, the biological mechanisms involved in attenuation are unknown. The objective of this study was to identify, through a comparative transcriptomics approach, the components of the B. bigemina virulent parasites that are differentially expressed in vivo, as opposed to those expressed by B. bigemina attenuated vaccine parasites when inoculated into naïve cattle. The biological material under study was obtained by inoculating spleen-intact cattle with infected erythrocytes containing either the attenuated strain or a virulent field strain. After RNA extraction, transcriptomic analysis (RNA-seq) was performed, followed by bioinformatic Differential Expression (DE) analysis and Gene Ontology (GO) term enrichment. The high-throughput sequencing results obtained by analyzing three biological replicates for each parasite strain ranged from 9,504,000 to 9,656,000, and 13,400,000 to 15,750,000 reads for the B. bigemina attenuated and virulent strains, respectively. At least 519 differentially expressed genes were identified in the analyzed strains. In addition, GO analysis revealed both similarities and differences across the three categories: cellular components, biological processes, and molecular functions. The attenuated strain of B. bigemina derived from in vitro culture presents global transcriptomic changes when compared to the virulent strain. Moreover, the obtained data provide insights into the potential molecular mechanisms associated with the attenuation or pathogenicity of each analyzed strain, offering molecular markers that might be associated with virulence or potential vaccine candidates.

Draft Genome Sequences of Mexican Babesia bovis Virulent and Attenuated Strains.

Babesia bovis, a tick-borne intraerythrocytic protozoan parasite that belongs to the phylum Apicomplexa, is one of the etiological agents of bovine babesiosis, a highly prevalent disease in tropical and subtropical countries that causes significant morbidity and deaths in cattle. This report presents the draft genome sequences of attenuated and virulent B. bovis strains of Mexican origin.

A Comparative Genomic Study of Attenuated and Virulent Strains of Babesia bigemina.

Cattle babesiosis is a socio-economically important tick-borne disease caused by Apicomplexa protozoa of the genus Babesia that are obligate intraerythrocytic parasites. The pathogenicity of Babesia parasites for cattle is determined by the interaction with the host immune system and the presence of the parasite's virulence genes. A Babesia bigemina strain that has been maintained under a microaerophilic stationary phase in in vitro culture conditions for several years in the laboratory lost virulence for the bovine host and the capacity for being transmitted by the tick vector. In this study, we compared the virulome of the in vitro culture attenuated Babesia bigemina strain (S) and the virulent tick transmitted parental Mexican B. bigemina strain (M). Preliminary results obtained by using the Basic Local Alignment Search Tool (BLAST) showed that out of 27 virulence genes described and analyzed in the B. bigemina virulent tick transmitted strain, only five were fully identified in the attenuated laboratory strain. In all cases, the identity and coverture of the identified genes of the wildtype strain were higher than those of the laboratory strain. This finding is putatively associated with the continuous partial loss of virulence genes in the laboratory strain after several passages of the parasite population under optimal in vitro growth conditions. The loss of virulence factors might be reflected in the absence of symptoms of the disease in cattle inoculated with the attenuated strain despite the presence of infection in the bovine host cells.

Challenges in Tick-Borne Pathogen Detection: The Case for Babesia spp. Identification in the Tick Vector.

The causative agents of Babesiosis are intraerythrocytic protozoa of the genus Babesia. Babesia parasites are present around the world, affecting several mammals including humans, pets and livestock, hence its medical and veterinary relevance. Babesia spp. detection in its invertebrate host is a main point in understanding the biology of the parasite to acquire more knowledge on the host-Babesia-vector interactions, as increasing knowledge of the Babesia lifecycle and babesiosis epidemiology can help prevent babesiosis outbreaks in susceptible mammals. The aim of the present review is to highlight the newest findings in this field, based on a bibliographic compilation of research studies recently carried out for the detection of the main Babesia species found in tick vectors affecting mammalian hosts, including the different tick stages such as adult ticks, larvae, nymphs and eggs, as well as the detection method implemented: microscopic tools for parasite identification and molecular tools for parasite DNA detection by conventional PCR, nested-PCR, PCR-RFLP, PCR-RLB hybridization, real time-PCR, LAMP and RAP assays. Although molecular identification of Babesia parasites has been achieved in several tick species and tissue samples, it is still necessary to carry out transmission experiments through biological models to confirm the vectorial capacity of various tick species.

An Overview of Current Knowledge on in vitro Babesia Cultivation for Production of Live Attenuated Vaccines for Bovine Babesiosis in Mexico.

The instrumentation of the in vitro culture system has allowed researchers to learn more about the metabolic and growth behavior of Babesia spp. The various applications for in vitro cultivation of Babesia include obtaining attenuated strains for vaccination or pre-munition, the selection of pure lines with different degrees of virulence, studies on biological cloning, ultrastructure, antigen production for diagnostics, drug sensitivity assessments, and different aspects of parasite biology. Although there are different types of vaccines that have been tested against bovine babesiosis, so far, the only procedure that has offered favorable results in terms of protection and safety has been the use of live attenuated vaccines. In countries, such as Australia, Argentina, Brazil, Uruguay and Israel, this type of vaccine has been produced and used. The alternative to live vaccines other than splenectomized calf-derived biological material, has been the in vitro cultivation of Babesia bovis and B. bigemina. The development of in vitro culture of Babesia spp. strains in a defined medium has been the basis for the initiation of a source of parasites and exoantigens for a variety of studies on the biochemistry and immunology of babesiosis. The use of live immunogens from attenuated strains derived from in vitro culture is highlighted, which has been proposed as an alternative to control bovine babesiosis. In several studies performed in Mexico, this type of immunogen applied to susceptible cattle has shown the induction of protection against the experimental heterologous strain challenge with both, Babesia-infected blood and animal exposure to confrontations on tick vector-infested farms. The combination of transfection technologies and the in vitro culture system as integrated methodologies would eventually give rise to the generation of genetically modified live vaccines. However, a greater challenge faced now by researchers is the large-scale cultivation of Babesia parasites for mass production and vaccine distribution.

Validation of an indirect ELISA using recombinant proteins as antigen to identify animals exposed to Babesia bigemina.

The objective of this study was to instrument a serological assay for the epidemiological diagnosis of bovine babesiosis in Mexico, using the Babesia bigemina recombinant protein RAP-1 (rRAP-1α) as antigen. rRAP-1α, r12d3 and rGP45 were the three recombinant antigens initially tested. Based on the highest titres obtained in the indirect ELISA (iELISA) with the positive control serum, using similar antigen concentrations, rRAP-1α was selected for further use. The diagnostic sensitivity and specificity rates estimated for the iELISA with rRAP-1α as antigen were 89.9% and 86.5%, respectively, while for the Indirect Fluorescent Antibody Test (IFAT), the gold standard assay, the sensitivity was 86.66% and the specificity was 95%. The ĸ agreement value determined was 0.52, indicating a moderate agreement between the iELISA and IFAT assays. The instrumented iELISA with rRAP-1α as antigen shows an excellent specificity rate and an acceptable sensitivity that allows for the detection of antibodies to B. bigemina in cattle naturally exposed to the vector tick Rhipicephalus microplus. By using the iELISA-rRAP-1α, along with an iELISA with recombinant Merozoite Surface Antigen (rMSA-1) for antibody determination against Babesia bovis in the serum samples collected from cattle at 'La Posta' experimental station in Mexico, a seroprevalence of 20.3% was estimated for B. bigemina and 19.4% for B. bovis, while 36.89% of samples were positive for both Babesia species. The iELISA test promises to be a safe and low-cost type of diagnosis available to cattle producers in Mexico and would facilitate the definition of herd immunity status to implement measures of control adapted for the prevention of bovine babesiosis outbreaks.

Diagnostic Tools for the Identification of Babesia sp. in Persistently Infected Cattle.

Bovine babesiosis is a tick-borne disease of cattle caused by the protozoan parasites of the genus Babesia. Babesia bovis, Babesia bigemina and Babesia divergens are considered by International health authorities (OIE) as the principal species of Babesia that cause bovine babesiosis. Animals that recover from a babesial primo infection may remain as persistent carriers with no clinical signs of disease and can be the source of infection for ticks that are able to acquire Babesia parasites from infected cattle and to transmit Babesia parasites to susceptible cattle. Several procedures that have been developed for parasite detection and diagnosis of this infectious carrier state constitute the basis for this review: A brief description of the direct microscopic detection of Babesia-infected erytrocytes; PCR-based diagnostic assays, which are very sensitive particularly in detecting Babesia in carrier cattle; in-vitro culture methods, used to demonstrate presence of carrier infections of Babesia sp.; animal inoculation, particularly for B. divergens isolation are discussed. Alternatively, persistently infected animals can be tested for specific antibabesial antibodies by using indirect serological assays. Serological procedures are not necessarily consistent in identifying persistently infected animals and have the disadvantage of presenting with cross reactions between antibodies to Babesia sp.

Using msa-2b as a molecular marker for genotyping Mexican isolates of Babesia bovis.

Variable merozoite surface antigens of Babesia bovis are exposed glycoproteins having a role in erythrocyte invasion. Members of this gene family include msa-1 and msa-2 (msa-2c, msa-2a(1), msa-2a(2) and msa-2b). To determine the sequence variation among B. bovis Mexican isolates using msa-2b as a genetic marker, PCR amplicons corresponding to msa-2b were cloned and plasmids carrying the corresponding inserts were purified and sequenced. Comparative analysis of nucleotide and deduced amino acid sequences revealed distinct degrees of variability and identity among the coding gene sequences obtained from 16 geographically different Mexican B. bovis isolates and a reference strain. Clustal-W multiple alignments of the MSA-2b deduced amino acid sequences performed with the 17 B. bovis Mexican isolates, revealed the identification of three genotypes with a distinct set each of amino acid residues present at the variable region: Genotype I represented by the MO7 strain (in vitro culture-derived from the Mexico isolate) as well as RAD, Chiapas-1, Tabasco and Veracruz-3 isolates; Genotype II, represented by the Jalisco, Mexico and Veracruz-2 isolates; and Genotype III comprising the sequences from most of the isolates studied, Tamaulipas-1, Chiapas-2, Guerrero-1, Nayarit, Quintana Roo, Nuevo Leon, Tamaulipas-2, Yucatan and Guerrero-2. Moreover, these three genotypes could be discriminated against each other by using a PCR-RFLP approach. The results suggest that occurrence of indels within the variable region of msa-2b sequences can be useful markers for identifying a particular genotype present in field populations of B. bovis isolated from infected cattle in Mexico.

Validation of a competitive enzyme-linked immunosorbent assay for detection of Babesia bigemina antibodies in cattle.

A competitive enzyme-linked immunosorbent assay (cELISA) based on a broadly conserved, species-specific, B-cell epitope within the C terminus of Babesia bigemina rhoptry-associated protein 1a was validated for international use. Receiver operating characteristic analysis revealed 16% inhibition as the threshold for a negative result, with an associated specificity of 98.3% and sensitivity of 94.7%. Increasing the threshold to 21% increased the specificity to 100% but modestly decreased the sensitivity to 87.2%. By using 21% inhibition, the positive predictive values ranged from 90.7% (10% prevalence) to 100% (95% prevalence) and the negative predictive values ranged from 97.0% (10% prevalence) to 48.2% (95% prevalence). The assay was able to detect serum antibody as early as 7 days after intravenous inoculation. The cELISA was distributed to five different laboratories along with a reference set of 100 defined bovine serum samples, including known positive, known negative, and field samples. The pairwise concordance among the five laboratories ranged from 100% to 97%, and all kappa values were above 0.8, indicating a high degree of reliability. Overall, the cELISA appears to have the attributes necessary for international application.

Primary midgut, salivary gland, and ovary cultures from Boophilus microplus.

Primary cell cultures from different tick organs are a valuable tool for host parasite research in the study of the protozoan Babesia sp., which infects different organs of the tick. In this work we describe the generation of midgut, salivary gland, and ovary primary cell cultures from dissections of Boophilus microplus. Midguts, salivary glands, and ovaries were dissected from B. microplus ticks on different days after bovine infestation; different enzymatic disaggregating protocols were tested in the presence of proteolytic enzymes, such as trypsin and collagenase type I and II, for tissue disaggregation and primary cell culture generation. The dissected tick organs obtained 18-20 days after bovine infestation showed a major cellular differentiation and were easier to identify by cellular morphology. The enzymatic disaggregation results showed that each tissue required a different proteolytic enzyme for optimal disaggregation; collagenase type I produced the most complete disaggregation for ovaries but not for midgut or salivary glands. Collagenase type II was effective for salivary glands but performed poorly on ovaries and midgets, and typsin was effective for midguts only. The midgut and ovary primary cell cultures were maintained for 4 weeks in optimal conditions after the cells were no longer viable. The salivary gland cell cultures were viable for 8 months.

Characterization of a Vitellogenin Gene Fragment in Boophilus microplus Ticks.

The objective of this study was to isolate, clone, and characterize a fragment of the vitellogenin (Vg) gene from a B. microplus tick strain from Mexico. Using cDNA and specific primers, an 1800-bp fragment was amplified, cloned, and transformed in into E. coli, and then sequenced. Comparative analysis with a previously reported sequence showed 99% identity at both the nucleotide and amino acid level. The predicted amino acid sequence of the Mexican Vg has 6 positive mutations. There is an insertion of an aspartic acid on position 26 and a deletion on position 552 with respect to the reported sequence. There were 11 predicted glycosylation sites conserved in both strains. It is concluded that there is a high sequence homology of Vg in both strains.

Phylogenetic analysis of Mexican Babesia bovis isolates using msa and ssrRNA gene sequences.

Variable merozoite surface antigens of Babesia bovis are exposed glycoproteins having a role in erythrocyte invasion. Members of this gene family include msa-1 and msa-2 (msa-2c, msa-2a(1), msa-2a(2), and msa-2b). Small subunit ribosomal (ssr)RNA gene is subject to evolutive pressure and has been used in phylogenetic studies. To determine the phylogenetic relationship among B. bovis Mexican isolates using different genetic markers, PCR amplicons, corresponding to msa-1, msa-2c, msa-2b, and ssrRNA genes, were cloned and plasmids carrying the corresponding inserts were sequenced. Comparative analysis of nucleotide and deduced amino acid sequences revealed distinct degrees of variability and identity among the coding gene sequences obtained from 12 geographically different B. bovis isolates and a reference strain. Overall sequence identities of 47.7%, 72.3%, 87.7%, and 94% were determined for msa-1, msa-2b, msa-2c, and ssrRNA, respectively. A robust phylogenetic tree was obtained with msa-2b sequences. The phylogenetic analysis suggests that Mexican B. bovis isolates group in clades not concordant with the Mexican geography. However, the Mexican isolates group together in an American clade separated from the Australian clade. Sequence heterogeneity in msa-1, msa-2b, and msa-2c coding regions of Mexican B. bovis isolates present in different geographical regions can be a result of either differential evolutive pressure or cattle movement from commercial trade.

Enhancement of the Mexican bovine babesiosis vaccine efficacy by using Lactobacillus casei.

To evaluate the effect of Lactobacillus casei on the effectiveness of the Mexican bovine babesiosis mixed vaccine, 20 bovines were randomly allocated into four groups of five animals (I, II, III, and IV). At day -2 animals in groups I and II were inoculated with saline solution by intramuscular route (i.m.) and animals in groups III and IV were inoculated with L. casei. At day 0 bovines in groups I and III were inoculated i.m. with bovine normal erythrocytes and animals of groups II and IV were inoculated with the babesiosis vaccine. Twenty-four days later each bovine was challenged with Babesia bovis- and B. bigemina-infected erythrocytes. The average rectal temperature in groups I and III was higher (P < 0.05) than that in the vaccinated groups after challenge. The average packed cell volume was lower (P < 0.01) in the control groups than in the vaccinated groups. At day 10 after challenge, the average anti-Babesia antibody level was higher in group IV than in group II. At day 7 after vaccination, the percentage of bovines positive to gamma interferon, as determined by real-time PCR, was 20, 0, 40, and 80 for groups I, II, III, and IV, respectively. All animals in control groups (I and III) were treated against babesiosis to avoid their death because they showed signs of babesiosis. The results indicate that L. casei, inoculated 2 days before the inoculation of the Mexican bivalent bovine babesiosis vaccine, improves the vaccine's efficiency.

Infection and seroconversion of susceptible animals introduced into a babesiosis endemic area.

The study aimed to determine the incubation period of Babesia sp. infection in naive cattle and to monitor the serological response once exposed to natural Boophilus microplus (Rhipicephalus microplus)-infested paddocks. The study was carried out on a farm located in Veracruz, Mexico. Five groups of five steers were relocated every 3 months from a tick-free area to a tick-infested paddock. Animals were introduced in October, January, April, July, and October. Blood samples were taken daily until day 21 to determine packed cell volume (PCV), percentage of parasitized erythrocytes (PPE), and antibody titers to Babesia bigemina and B. bovis by the indirect fluorescent antibody procedure. Detection of Babesia in blood was also performed by species-specific PCR. The estimated incubation period was 6-14 days post introduction to paddocks (PIP), with fever (41 degrees C) for at least 3 days. PCV decreased by >25% and Babesia parasites were observed during the clinical phase of the disease. The highest individual PPEs (0.44% and 0.22% for B. bovis and B. bigemina, respectively) were observed from animals introduced in October. The four other groups showed a mean PPE ranging from 0.002-0.146% at day 14 PIP. All animals were detected as PCR positive between 8-14 days PIP. The highest antibody titers were 1:3328. The environmental conditions were favorable throughout the year for tick reproduction as the farm showed enzootic stability and hyperendemic conditions for bovine babesiosis. In this type of farm, strategic tick control could be accompanied by babesiosis vaccination, particularly for cattle relocated from tick-free areas.

Expression analysis of heat shock protein 20 and rhoptry-associated protein 1a in sexual stages and kinetes of Babesia bigemina.

Heat shock protein 20 (HSP-20) and rhoptry-associated protein 1a (RAP-1a) are two proteins considered as candidates to be included in vaccines or diagnostics methods for the control of bovine babesiosis. It has been hypothesized that both genes have a basic function in the cellular physiology of erythrocyte-infecting stages; it is not known if they have a functional role in tick stages. The objective of this work was to analyze whether hsp-20 and rap-1a are expressed in sexual stages and kinetes of Babesia bigemina. Purified RNA from sexual stages and kinetes was analyzed by reverse transcriptase (RT)-PCR with specific primers for hsp-20 or rap-1a. Expression analysis was carried out using an indirect immunofluorescence test with specific antibodies against HSP-20 and RAP-1a. Results obtained by RT-PCR showed amplicons for hsp-20 and rap-1a in sexual stages and kinetes. Positive signals were also detected when sexual stages and kinetes were analyzed with specific antibodies for HSP-20 and RAP-1a. The results obtained here confirm the hypothesis that the genes hsp-20 and rap-1a from B. bigemina are expressed in sexual stages and kinetes and stress the importance of these proteins in the cellular physiology of tick stages.

msa-1 and msa-2c gene analysis and common epitopes assessment in Mexican Babesia bovis isolates.

Babesia bovis msa-1 and msa-2c genes belong to the variable merozoite surface antigen gene family. These genes code for antigenic proteins present on the merozoite surface (MSA) and are involved in the parasite invasion to the bovine erythrocyte. Previous studies carried out on MSA-1 have evidenced antigen allelic variation in B. bovis isolates from similar endemic regions, as well as in isolates from different geographic regions of the world (Argentina, Australia, Israel). Studies conducted on MSA-2c, however, have shown that this antigen is widely conserved on isolates from distinct geographic regions. In this study, it was hypothesized that MSA-1 and MSA-2c antigens would contain common epitopes despite the presence of nucleotide sequence differences found in 13 B. bovis isolates and strains collected in geographically distant regions of Mexico. Bioinformatics analysis of the primary structure from DNA fragments derived from PCR amplification, cloning, and sequencing of msa-1 and msa-2c genes from the 13 B. bovis populations revealed that the msa-1 gene product present in the various isolates tested is less conserved among isolates obtained within a similar geographic region in Mexico (51-99.7% sequence identity). Results obtained by immunoblot analysis of B. bovis protein extracts reacted with a monoclonal antibody to MSA-1 42-kDa antigen, conclusively showed cross-reactive common epitopes only in Mexican isolates having high sequence identity (>/=99%, eight isolates). Sequence analysis and multiple alignment of deduced MSA-2c demonstrated a high degree of sequence identity (90-100%) among the Mexican B. bovis isolates and strains. Immunoblot results using a polyclonal antibody to MSA-2c reacted against the protein extracts recognized conserved epitopes in at least nine of the B. bovis isolates. The results obtained in this study agree with those previously reported by other researchers and confirm that, based in sequence identity conservation in Mexican B. bovis isolates and strains so far collected and analyzed, MSA-2c represents an ideal antigen worth evaluating as a vaccine candidate.

Identification of common antigens in Babesia bovis, B. bigemina, and B. divergens.

Bovine babesiosis, caused by Babesia bovis, B. bigemina, and B. divergens, is a significant impediment to livestock production in countries with tropical/subtropical and temperate climates. Previous studies conducted on the immunoprophylaxis against the disease and diagnosis of these parasites has demonstrated the presence of similar antigens. The objective of this article was to identify and partially characterize antigens conserved among these three species. Immunochemical analysis using sera from cattle immunized individually with antigens from these three Babesia species revealed a number of antigens recognized by heterologous antisera. Cross-reactions were more evident in sera from cattle immunized with B. bovis/B. bigemina which recognized several antigens (15 kDa to >200 kDa) in B. divergens. Immunoscreening of a B. divergens cDNA library with bovine serum to B. bigemina allowed the isolation of five clones and DNA sequencing of plasmid BdJF5 showed a 680 bp cDNA insert. Basic Local Alignment Search Tool (BLAST) analysis of the predicted amino acid sequence revealed 47% identity with a protein identified as alphaNAC. Serum from mice immunized with a recombinant Glutathione S-Transferase-BdJF5 fusion protein immunoprecipitated a 20 kDa B. bovis antigen. However, 30 kDa and 18 kDa antigens were immunoprecipitated from B. divergens and immunoblotting analysis revealed the recognition of a 35 kDa B. bigemina antigen. An indirect fluorescence antibody assay on merozoites showed strong reaction with B. divergens and weak recognition of B. bovis and B. bigemina. Despite the existent antigenic polymorphism among the Babesia spp., these results demonstrated that common antigens occur between European B. divergens and Mexican B. bovis/B. bigemina.

Evaluation of cattle inoculated with Babesia bovis clones adhesive in vitro to bovine brain endothelial cells.

A comparative assessment of the virulence of Babesia bovis clones that adhere or not to bovine brain endothelial cells was done using two clones of B. bovis: (1) a clone phenotypically characterized as virulent (2F8) and (2) a clone of reduced virulence (RAD). Of these subpopulations, we selected those that had adhesive characteristics (a) or nonadhesive characteristics (na) in cultured endothelial cells. Twenty Holstein cattle, 12 months of age or older, were used in this study, and these cattle were randomly assigned to five groups of four animals each. The clones and their respective subpopulations were inoculated via intramuscular injection at a 0.5 x 10(7) infected erythrocyte dosage. Group A was inoculated with aRAD, group B with naRAD, group C with a2F8, group D with na2F8, and group E remained as a control. All inoculated animals showed a decrease in the packed cell volume (PCV), with group D showing the largest decrease (39.53%) and longest time (7 days) with rectal temperature above 39.5 degrees C. Babesia was observed in stained blood smears from only six cattle. While the four parasite subpopulations were pathogenic, significant differences were not noted among them, despite that the subpopulations considered to be virulent caused the greatest reduction in PCV per individual.

Bovine babesiosis live vaccine production: use of gamma irradiation on the substrate.

Gamma irradiation on bovine serum and red blood cells (RBC) allows proliferation and growth of in vitro-cultured Babesia sp., and has potential application to inactivate contaminating viruses and bacteria from the substrate. Gamma irradiation with 25 kGy in a source of (60)Co was able to inactivate infectious bovine rinotracheitis (IBR) and bovine viral diarrhea (BVD) viruses in artificially contaminated serum; besides, bacteria were also eliminated. In vitro culture of Babesia bovis (B. bovis) in modified substrate, by adding irradiated serum with (60)Co at 25 kGy was propagated from 24-well culture plates to 225 cm(2) tissue culture flasks, and percentages of parasitized erythrocytes (PPE) from 2.4% to 8.8% were obtained. Infected RBC adapted to Irrad S were transferred to the irradiated substrate in vitro culture system, by using serum irradiated at 25 kGy and RBC from 10 to 70 Gy. The PPE ranged from 3.1 to 11. Culture of Babesia bigemina (B. bigemina) was established with Irrad S (25 kGy); its propagation was achieved in tissue culture flasks reaching PPE from 0.5 to 4.3 with no statistical difference (P > 0.05) when compared to the nonirradiated control culture (1.2-4.8). B. bigemina-infected RBCs were transferred to the modified culture system by adding irradiated serum and RBC (25 kGy and 70 Gy, respectively). PPE obtained in culture flasks were from 0.8 to 4.2. The results indicate that gamma irradiation is a suitable method to inactivate potential viral contamination and eliminate bacteria from bovine serum, to produce a live attenuated vaccine through the in vitro culture.

Identification of a coronin-like protein in Babesia species.

The present study was designed to immunochemically identify a coronin-like protein in Babesia bovis, B. bigemina, B. divergens, and B. canis. A 2-kbp cDNA insert of B. bovis carried by plasmid BvN9 was sequenced by the dideoxichain-termination method on both strands. The cDNA insert contained a 1719-bp long open reading frame coding for a deduced protein sequence of 61.7 kDa. Sequence analysis using the PSI-BLAST program revealed about 30% protein sequence identity with a coronin-like protein of Plasmodium falciparum. The encoding sequence of the cDNA insert lacking 70 amino acids at the N-terminal was subcloned in frame into pGEX 4T-3 to produce a recombinant glutathione S-transferase (GST)-pBv fusion protein. Polyclonal antibodies prepared in rabbits immunized with the purified GST-fusion protein recognized a Babesia-specific component of approximately 60 kDa by immunoprecipitation with [35S]methionine-labeled parasites. However, two molecules with relative sizes of 60 and 70 kDa were recognized in Babesia-infected erythrocyte extracts by immunobloting analysis. The 70-kDa component was apparently of host erythrocyte origin. In an indirect fluorescent antibody test, the rabbit serum strongly reacted with the merozoite stage of the four Babesia species, but also, although weakly, with the host erythrocyte. A cosedimentation assay performed with GST-pBv fusion protein and exogenous actin from rabbit liver showed that the GST-pBv fusion protein, but not the GST protein, was associated to actin. From these results, we conclude that the protein present in the four Babesia species analyzed here may be considered as a novel coronin-like, actin-binding protein.