Development and standardization of a Loop-mediated isothermal amplification (LAMP) test for the detection of Babesia bigemina.

Bovine babesiosis is a tick-borne disease caused by protozoan parasites of the genus Babesia. Babesia bigemina is one of the most prevalent and economically important parasite species that infects cattle because of its impact on the meat and milk production industry. Effective disease control strategies should include detection of reservoir animals and early and specific pathogen detection using rapid, economical, sensitive, and specific detection techniques. The loop-mediated isothermal amplification technique (LAMP) is a one-step molecular reaction that amplifies DNA sequences with high sensitivity and specificity under isothermal conditions and requires no special equipment. The results can be observed by the naked eye as color changes. The aim of this work was to develop and standardize the LAMP technique for B. bigemina detection and its visualization using hydroxynaphtol blue. For this situation, primers were designed from the conserved sequences of the B. bigemina ama-1 gene. The results showed that at 63 °C in 1 h and under standardized conditions, this technique could amplify B. bigemina DNA as indicated by the characteristic colorimetric change. Sensitivity evaluation indicated that DNA was amplified at a 0.00000001% parasitemia, and it was demonstrated that this technique specifically amplified the DNA of B. bigemina. Additionally, this technique could amplify DNA from 10 strains of B. bigemina from three different countries. It is concluded that the LAMP technique as modified in our case could specifically amplify B. bigemina DNA and shows high sensitivity, does not cross-react with related organisms, and the product is observed by 60 min of reaction time based on color changes. This report is the first LAMP report that uses sequences that are conserved between strains of the ama-1 gene, demonstrates the results by color changes using hydroxynaphtol blue. We propose LAMP as a rapid and economical alternative method for the molecular detection of B. bigemina.

Thrombospondin Related Anonymous Protein Superfamily in Vector-Borne Apicomplexans: The Parasite's Toolkit for Cell Invasion.

Apicomplexan parasites transmitted by vectors, including Babesia spp. and Plasmodium spp., cause severe disease in both humans and animals. These parasites have a complex life cycle during which they migrate, invade, and replicate in contrasting hosts such as the mammal and the invertebrate vector. The interaction of parasites with the host cell is mediated by adhesive proteins which play a key role in the different cellular processes regarding successful progression of the life cycle. Thrombospondin related anonymous protein (TRAP) is a superfamily of adhesins that are involved in motility, invasion and egress of the parasite. These proteins are stored and released from apical organelles and have either one or two types of adhesive domains, namely thrombospondin type 1 repeat and von Willebrand factor type A, that upon secretion are located in the extracellular portion of the molecule. Proteins from the TRAP superfamily have been intensively studied in Plasmodium species and to a lesser extent in Babesia spp., where they have proven to be functionally relevant throughout the entire parasite's journey both in the arthropod vector and in the mammalian host. In recent years new findings provided answers to the role of TRAP proteins and in some cases the function of these adhesins during the parasite's life cycle was redefined. In this review we will discuss the current knowledge of the diverse roles of the TRAP superfamily in vector-borne parasites from Class Aconoidasida. We will focus on the varied approaches that allowed the understanding of protein function and the relevance of TRAP- superfamily throughout the entire parasite's cell cycle.

Babesia bovis AMA-1, MSA-2c and RAP-1 contain conserved B and T-cell epitopes, which generate neutralizing antibodies and a long-lasting Th1 immune response in vaccinated cattle.

Vaccines against bovine babesiosis must, ideally, induce a humoral immune response characterized by neutralizing antibodies against conserved epitopes and a cellular Th1 immune response. In Babesia bovis, proteins such as AMA-1, MSA-2c, and RAP-1 have been characterized and antibodies against these proteins have shown a neutralizing effect, demonstrating the implication of B and T-cell epitopes in the immune response. There is evidence of the existence of B and T-cell epitopes in these proteins, however, it remains to be defined, the presence of conserved peptides in strains from around the world containing B and T-cell epitopes, and their role in the generation of a long-lasting immunity. The aim in this paper was to identify peptides of Babesia bovis AMA-1, MSA-2c, and RAP-1 that elicit a neutralizing and long-lasting Th1 immune response. Peptides containing B-cell epitopes of AMA-1, MSA-2c and RAP-1, were identified. The immune response generated by each peptide was characterized in cattle. All peptides tested induced antibodies that recognized intraerythrocytic parasites, however, only 5 peptides generated neutralizing antibodies in vitro: P2AMA-1 (6.28%), P3MSA-2c (10.27%), P4MSA-2c (10.42%), P1RAP-1 (32.45%), and P4RAP-1 (36.98%). When these neutralizing antibodies were evaluated as a pool, the inhibition percentage of invasion increased to 52.37%. When the T cellular response was evaluated, two peptides: P3MSA2c and P2AMA1 induced a higher percentage (>70%) of activated CD4 +/CD45RO+ T cells than unstimulated cells. Additionally, both peptides induced the production of gamma interferon (IFN-) in PBMCs from vaccinated cattle after one year proving the implication of a long-lasting Th1 immune response. In conclusion, we identified conserved peptides containing B and T-cell epitopes in antigens of B. bovis that elicit a Th1 immune response and showed evidence that peptides from the same protein elicit different immune responses, which has implication for vaccine development in bovine babesiosis.

Diversity of the BoLA-DRB3 gene in cattle breeds from tropical and subtropical regions of Argentina.

Bovine leukocyte antigens (BoLA) have been widely studied because of their primary function in the recognition of pathogens by the immune system. To date, however, the characterization of the BoLA-DRB3 gene in Latin American Zebu and mixed zebuine breeds is scarce. By a sequence-based typing method, here we sequenced exon 2 of BoLA class II DRB3 gene in 264 animals from the five most commonly used breeds in northern Argentina (Creole, Brahman, Braford, Brangus, and Nellore).The Bos taurus, Bos indicus, and mixed breeds analyzed here contained 61 previously reported alleles. Genetic diversity was high at both allelic and nucleotide sequence levels, particularly in the mixed breeds Braford and Brangus. In contrast to previous reports on DRB3 diversity, no evidence of balancing selection was found in our data. Differentiation among breeds was highly significant, as shown by FST (FST = 0.052, P < 0.001) and cluster analyses. In accordance with historical origin of the breeds, UPGMA trees and metric multidimensional scaling (MDS) analyses showed that Creole is distantly related to the other zebuine breeds. Among them, Brahman, Braford, and Brangus exhibited the closest affiliations. Despite the overall differentiation of the breeds, analysis of the peptide binding regions at the aminoacid level revealed that the key aminoacids involved in peptide recognition are greatly conserved suggesting little influence of domestication and breeding in functional MHC variability. In sum, this is the first report of BoLA-DRB3 diversity in pure and mixed Bos indicus cattle breeds from Argentina. Knowledge of BoLA-DRB3 variability in breeds adapted to tropical and subtropical environments contributes not only to the characterization of MHC diversity but also to the design of peptide-based vaccines.

Development of highly sensitive one step-PCR tests for improved detection of B. bigemina and B. bovis.

Bovine babesiosis caused by Babesia bigemina and B. bovis is an economically relevant tick-borne disease distributed over tropical and subtropical world regions. Animals that recover from the clinical disease can remain persistently infected, and those carriers are epidemiologically relevant since they can act as a source of infection to other animals through the tick bite. According to the manual of the World Organisation for Animal Health (OIE), the recommended molecular diagnosis test for both parasites is a nested polymerase chain reaction (nPCR) based on an amplification of a fragment of the rap-1 gene. Since nPCRs are time consuming, have a higher cost and risk of contamination, we propose a single step PCR for B. bigemina (BbiVESA) and B. bovis (BboVESA) based on the amplification of the multi-copy ves-1α gene. We developed these methods and we achieved a detection limit of 1 × 10-12 % parasitemia for B. bigemina and of 1 × 10-6 % for B. bovis using reference strains, which compared to the reference OIE tests, results in an improvement in sensitivity of six orders for B. bigemina. Finally, we tested 48 field samples from a babesiosis enzootic region where we were able to detect a higher proportion of positive animals with both VESA methods than with the reference rap-1 nPCRs. This difference was statistically significant for each Babesia species. Concordance between both diagnostic schemes based on Cohen's kappa coefficient showed minimal to non-agreement (κ = 0.32) for B. bigemina and non-agreement (κ = 0.16) for B. bovis since BbiVESA and BboVESA PCR tests showed a significantly higher detection capacity. In conclusion, the high sensitivity of the assay, together with the lower demand of time and reagents make the VESA PCR methods developed here a valuable diagnostic tool for the molecular detection and epidemiological survey of both Babesia pathogens.

The key to egress? Babesia bovis perforin-like protein 1 (PLP1) with hemolytic capacity is required for blood stage replication and is involved in the exit of the parasite from the host cell.

Bovine babesiosis is a tick-borne disease caused by apicomplexan parasites of the Babesia genus that represents a major constraint to livestock production worldwide. Currently available vaccines are based on live parasites which have archetypal limitations. Our goal is to identify candidate antigens so that new and effective vaccines against Babesia may be developed. The perforin-like protein (PLP) family has been identified as a key player in cell traversal and egress in related apicomplexans and it was also identified in Babesia, but its function in this parasite remains unknown. The aim of this work was to define the PLP family in Babesia and functionally characterize PLP1, a representative member of the family in Babesia bovis. Bioinformatic analyses demonstrate a variable number of plp genes (four to eight) in the genomes of six different Babesia spp. and conservation of the family members at the secondary and tertiary structure levels. We demonstrate here that Babesia PLPs contain the critical domains present in other apicomplexan PLPs to display the lytic capacity. We then focused on the functional characterization of PLP1 of B. bovis, both in vitro and in vivo. PLP1 is expressed and exposed to the host immune system during infection and has high hemolytic capacity under a wide range of conditions in vitro. A B. bovis plp1 knockout line displayed a decreased growth rate in vitro compared with the wild type strain and a peculiar phenotype consisting of multiple parasites within a single red blood cell, although at low frequency. This phenotype suggests that the lack of PLP1 has a negative impact on the mechanism of egression of the parasite and, therefore, on its capacity to proliferate. It is possible that PLP1 is associated with other proteins in the processes of invasion and egress, which were found to have redundant mechanisms in related apicomplexans. Future work will be focused on unravelling the network of proteins involved in these essential parasite functions.

Identification and characterization of a Babesia bigemina thrombospondin-related superfamily member, TRAP-1: a novel antigen containing neutralizing epitopes involved in merozoite invasion.

BACKGROUND: Thrombospondin-related anonymous protein (TRAP) has been described as a potential vaccine candidate for several diseases caused by apicomplexan parasites. However, this protein and members of this family have not yet been characterized in Babesia bigemina, one of the most prevalent species causing bovine babesiosis. METHODS: The 3186-bp Babesia bigemina TRAP-1 (BbiTRAP-1) gene was identified by a bioinformatics search using the B. bovis TRAP-1 sequence. Members of the TRAP and TRAP-related protein families (TRP) were identified in Babesia and Theileria through a search of the TSP-1 adhesive domain, which is the hallmark motif in both proteins. Structural modeling and phylogenetic analysis were performed with the identified TRAP proteins. A truncated recombinant BbiTRAP-1 that migrates at approximately 107 kDa and specific antisera were produced and used in Western blot analysis and indirect fluorescent antibody tests (IFAT). B-cell epitopes with neutralizing activity in BbiTRAP-1 were defined by enzyme-linked immunosorbent assays (ELISA) and invasion assays. RESULTS: Three members of the TRAP family of proteins were identified in B. bigemina (BbiTRAP-1 to -3). All are type 1 transmembrane proteins containing the von Willebrand factor A (vWFA), thrombospondin type 1 (TSP-1), and cytoplasmic C-terminus domains, as well as transmembrane regions. The BbiTRAP-1 predicted structure also contains a metal ion-dependent adhesion site for interaction with the host cell. The TRP family in Babesia and Theileria species contains the canonical TSP-1 domain but lacks the vWFA domain and together with TRAP define a novel gene superfamily. A variable number of tandem repeat units are present in BbiTRAP-1 and could be used for strain genotyping. Western blot and IFAT analysis confirmed the expression of BbiTRAP-1 by blood-stage parasites. Partial recognition by a panel of sera from B. bigemina-infected cattle in ELISAs using truncated BbiTRAP-1 suggests that this protein is not an immunodominant antigen. Additionally, bovine anti-recombinant BbiTRAP-1 antibodies were found to be capable of neutralizing merozoite invasion in vitro. CONCLUSIONS: We have identified the TRAP and TRP gene families in several Babesia and Theileria species and characterized BbiTRAP-1 as a novel antigen of B. bigemina. The functional relevance and presence of neutralization-sensitive B-cell epitopes suggest that BbiTRAP-1 could be included in tests for future vaccine candidates against B. bigemina.

Immunisation of cattle against Babesia bovis combining a multi-epitope modified vaccinia Ankara virus and a recombinant protein induce strong Th1 cell responses but fails to trigger neutralising antibodies required for protection.

Protection against the intraerythrocytic protozoan parasite Babesia bovis depends on both strong innate and adaptive immune response, this latter involving the presentation of parasite antigens to CD4+ T-lymphocytes by professional antigen-presenting cells. Secretion of Th1 cytokines by CD4+ T cell is also very important for isotype switching to IgG2, the best opsonising antibody isotype in cattle, to target extracellular parasites and parasite antigens displayed at the erythrocyte surface. In the field of vaccinology, heterologous prime-boost schemes combining protein-adjuvant formulations with a modified vaccinia Ankara vector expressing the same antigen have demonstrated the induction of both humoral and cellular immune responses. It has been previously demonstrated that MVA-infected dendritic cells can present antigens in the context of MHC II and activate CD4+ T cell. These results support the use of the MVA viral vector for a pathogen like Babesia bovis, which only resides within erythrocytes. In this study, 13-15-months-old Holstein-Friesian steers were immunised with a subunit vaccine as a prime and a modified vaccinia Ankara vector as a boost, both expressing a chimeric multi-antigen (rMABbo - rMVA). This antigen includes the immunodominant B and T cell epitopes of three B. bovis proteins: merozoite surface antigen - 2c (MSA - 2c), rhoptry associated protein 1 (RAP - 1) and heat shock protein 20 (HSP20). Responses were compared with the Babesia bovis live attenuated vaccine used in Argentina (R1A). Eleven weeks after the first immunisation, all bovines were challenged by the inoculation of a virulent B. bovis strain. All groups were monitored daily for hyperthermia and reduction of packed cell volume. Both the rMABbo - rMVA and R1A vaccinated animals developed high titters of total IgG antibodies and an antigen-specific Th1 cellular response before and after challenge. However, all rMABbo - rMVA steers showed clinical signs of disease upon challenge. Only the R1A live vaccine group developed an immune response associated with in vitro neutralising antibodies at a level that significantly inhibited the parasite invasion. The lack of protection observed with this recombinant formulation indicates the need to perform further basic and clinical studies in the bovine model in order to achieve the desired effectiveness. This is the first report in which a novel vaccine candidate against Babesia bovis was constructed based on a recombinant and rationally designed viral vector and evaluated in the biological model of the disease.

Development of an Indirect ELISA Based on a Recombinant Chimeric Protein for the Detection of Antibodies against Bovine Babesiosis.

The current method for Babesia spp. serodiagnosis based on a crude merozoite antigen is a complex and time-consuming procedure. An indirect enzyme-linked immunosorbent assay (iELISA) based on a recombinant multi-antigen of Babesia bovis (rMABbO) was developed for detection of antibodies in bovines suspected of infection with this parasite. The multi-antigen comprises gene fragments of three previously characterized B. bovis antigens: MSA-2c, RAP-1 and the Heat Shock protein 20 that are well-conserved among geographically distant strains. The cutoff value for the new rMABbo-iELISA was determined using 75 known-positive and 300 known-negative bovine sera previously tested for antibodies to B. bovis by the gold-standard ELISA which uses a merozoite lysate. A cutoff value of ≥35% was determined in these samples by receiver operator characteristic (ROC) curve analysis, showing a sensitivity of 95.9% and a specificity of 94.3%. The rMABbo-iELISA was further tested in a blind trial using an additional set of 263 field bovine sera from enzootic and tick-free regions of Argentina. Results showed a good agreement with the gold standard test with a Cohen's kappa value of 0.76. Finally, the prevalence of bovine babesiosis in different tick enzootic regions of Argentina was analyzed where seropositivity values among 68-80% were obtained. A certain level of cross reaction was observed when samples from B. bigemina infected cattle were analyzed with the new test, which can be attributed to shared epitopes between 2 of the 3 antigens. This new rMABbo-iELISA could be considered a simpler alternative to detect anti Babesia spp. antibodies and appears to be well suited to perform epidemiological surveys at the herd level in regions where ticks are present.

Epidemiology of Babesia, Anaplasma and Trypanosoma species using a new expanded reverse line blot hybridization assay.

Vector-borne hemoparasitic infections are a major problem that affects livestock industries worldwide, particularly in tropical and subtropical regions. In this work, a reverse line blot (RLB) hybridization assay was developed for the simultaneous detection and identification of Anaplasma, Babesia and bovine trypanosomes, encompassing in this way the most relevant hemoparasites that affect cattle. A total of 186 bovine blood samples collected from two different ecoepidemiological regions of northeast Argentina, with and without tick control, were analyzed with this new RLB. High diversity of parasites, such as Babesia bovis, B. bigemina, Anaplasma marginale and three different Trypanosoma species, was found. High rates of coinfections were also detected, and significant differences were observed not only in the prevalence of parasites but also in the level of coinfections between the two analyzed areas. Regarding the Trypanosoma genus, we provide molecular evidence of the presence of T. vivax and T. theileri for the first time in Argentina. Besides, since the RLB is a prospective tool, it allowed the identification of a yet unknown bovine trypanosome which could not be assigned to any of the bovine species known so far. In the present study we provide new insights on the prevalence of several pathogens that directly impact on livestock production in Argentina. The RLB assay developed here allows to identify simultaneously numerous pathogenic species which can also be easily expanded to detect other blood borne pathogens. These characteristics make the RLB hybridization assay an essential tool for epidemiological survey of all vector-borne pathogens.

Evaluation of different heterologous prime-boost immunization strategies against Babesia bovis using viral vectored and protein-adjuvant vaccines based on a chimeric multi-antigen.

Protection against the intraerythrocytic bovine parasite Babesia bovis requires both humoral and cellular immune responses. Therefore, tailored combinations of immunogens targeted at both arms of the immune system are strategies of choice to pursue sterilizing immunity. In this study, different heterologous prime-boost vaccination schemes were evaluated in mice to compare the immunogenicity induced by a recombinant adenovirus, a modified vaccinia Ankara vector or a subunit vaccine all expressing a chimeric multi-antigen. This multi-antigen includes the immunodominant B and T cell epitopes of three B. bovis proteins: Merozoite Surface Antigen - 2c (MSA-2c), Rhoptry Associated Protein - 1 (RAP-1) and Heat Shock Protein 20 (HSP20). Both priming with the adenovirus or recombinant multi-antigen and boosting with the modified vaccinia Ankara vector achieved a high degree of activation of TNFα and IFNγ-secreting CD4(+) and CD8(+) specific T cells 60days after the first immunization. High titers of specific IgG antibodies were also detected at the same time point and lasted up to day 120 of the first immunization. Only the adenovirus - MVA combination triggered a marked isotype skew for the IgG2a antibody subclass meanwhile for the other immune traits analyzed here, both vaccination schemes showed similar performances. The immunological characterization in the murine model of these rationally designed immunogens led us to propose that adenoviruses as well as the bacterially expressed multi-antigen are highly reliable primer candidates to be considered in future experiments in cattle to test protection against bovine babesiosis.

Molecular identification of Anaplasma marginale in two autochthonous South American wild species revealed an identical new genotype and its phylogenetic relationship with those of bovines.

BACKGROUND: Anaplasma marginale is a well-known cattle pathogen of tropical and subtropical world regions. Even though, this obligate intracellular bacterium has been reported in other host species different than bovine, it has never been documented in Myrmecophaga tridactyla (giant anteater) or Hippocamelus antisense (taruca), which are two native endangered species. METHODS: Samples from two sick wild animals: a Myrmecophaga tridactyla (blood) and a Hippocamelus antisense (blood and serum) were studied for the presence of A. marginale DNA through msp5 gene fragment amplification. Further characterization was done through MSP1a tandem repeats analysis and MLST scheme and the genetic relationship among previously characterized A. marginale sequences were studied by applying, eBURST algorithm and AMOVA analysis. RESULTS: Anaplasma marginale DNA was identified in the Myrmecophaga tridactyla and Hippocamelus antisense samples. Through molecular markers, we identified an identical genotype in both animals that was not previously reported in bovine host. The analysis through eBURST and AMOVA revealed no differentiation between the taruca/anteater isolate and the bovine group. CONCLUSIONS: In the present publication we report the identification of A. marginale DNA in a novel ruminant (Hippocamelus antisense) and non-ruminant (Myrmecophaga tridactyla) host species. Genotyping analysis of isolates demonstrated the close relatedness of the new isolate with the circulation population of A. marginale in livestock. Further analysis is needed to understand whether these two hosts contribute to the anaplasmosis epidemiology.

Development of a multilocus sequence typing scheme for the study of Anaplasma marginale population structure over space and time.

Bovine Anaplasmosis caused by Anaplasma marginale is a worldwide disease prevalent in tropical and subtropical regions where Rhipicephalus microplus is considered the most significant biological vector. Molecular markers previously applied for A. marginale typing are efficient for isolate discrimination but they are not a suitable tool for studying population structure and dynamics. Here we report the development of an MLST scheme based on the study of seven genes: dnaA, ftsZ, groEl, lipA, recA, secY and sucB. Five annotated genomes (Saint Maries, Florida, Mississippi, Puerto Rico and Virginia) and 53 bovine blood samples from different world regions were analyzed. High nucleotide diversity and a large proportion of synonymous substitutions, indicative of negative selection resulted from DnaSP 5.00.02 package application. Recombination events were detected in almost all genes, this evidence together with the coexistence of more than one A. marginale strain in the same sample might suggest the superinfection phenomena as a potential source of variation. The allelic profile analysis performed through GoeBURST shown two main CC that did not support geography. In addition, the AMOVA test confirmed the occurrence of at least two main genetically divergent groups. The composition of the emergent groups reflected the impact of both historical and environmental traits on A. marginale population structure. Finally, a web-based platform "Galaxy MLST-Pipeline" was developed to automate DNA sequence editing and data analysis that together with the Data Base are freely available to users. The A. marginale MLST scheme developed here is a valuable tool with a high discrimination power, besides PCR based strategies are still the better choice for epidemiological intracellular pathogens studies. Finally, the allelic profile describe herein would contribute to uncover the mechanisms in how intracellular pathogens challenge virulence paradigm.

A Neospora caninum vaccine using recombinant proteins fails to prevent foetal infection in pregnant cattle after experimental intravenous challenge.

The aim of the present study was to evaluate the immunogenicity and protective efficacy of rNcSAG1, rNcHSP20 and rNcGRA7 recombinant proteins formulated with immune stimulating complexes (ISCOMs) in pregnant heifers against vertical transmission of Neospora caninum. Twelve pregnant heifers were divided into 3 groups of 4 heifers each, receiving different formulations before mating. Immunogens were administered twice subcutaneously: group A animals were inoculated with three recombinant proteins (rNcSAG1, rNcHSP20, rNcGRA7) formulated with ISCOMs; group B animals received ISCOM-MATRIX (without antigen) and group C received sterile phosphate-buffered saline (PBS) only. The recombinant proteins were expressed in Escherichia coli and purified nickel resin. All groups were intravenously challenged with the NC-1 strain of N. caninum at Day 70 of gestation and dams slaughtered at week 17 of the experiment. Heifers from group A developed specific antibodies against rNcSAG1, rNcHSP20 and rNcGRA7 prior to the challenge. Following immunization, an statistically significant increase of antibodies against rNcSAG1 and rNcHSP20 in all animals of group A was detected compared to animals in groups B and C at weeks 5, 13 and 16 (P<0.001). Levels of antibodies against rNcGRA7 were statistical higher in group A animals when compared with groups B and C at weeks 5 and 16 (P>0.001). There were no differences in IFN-γ production among the experimental groups at any time point (P>0.05). Transplacental transmission was determined in all foetuses of groups A, B and C by Western blot, immunohistochemistry and nested PCR. This work showed that rNcSAG1, rNcHSP20 and rNcGRA7 proteins while immunogenic in cattle failed to prevent the foetal infection in pregnant cattle challenged at Day 70 of gestation.

Vaccine strategies against Babesia bovis based on prime-boost immunizations in mice with modified vaccinia Ankara vector and recombinant proteins.

In this study, a recombinant modified vaccinia virus Ankara vector expressing a chimeric multi-antigen was obtained and evaluated as a candidate vaccine in homologous and heterologous prime-boost immunizations with a recombinant protein cocktail. The chimeric multi-antigen comprises immunodominant B and T cell regions of three Babesia bovis proteins. Humoral and cellular immune responses were evaluated in mice to compare the immunogenicity induced by different immunization schemes. The best vaccination scheme was achieved with a prime of protein cocktail and a boost with the recombinant virus. This scheme induced high level of specific IgG antibodies and secreted IFN and a high degree of activation of IFNγ(+) CD4(+) and CD8(+) specific T cells. This is the first report in which a novel vaccine candidate was constructed based on a rationally designed multi-antigen and evaluated in a prime-boost regime, optimizing the immune response necessary for protection against bovine babesiosis.

Immunogenicity of hypothetical highly conserved proteins as novel antigens in Anaplasma marginale.

Anaplasma marginale is a tick-transmitted Gram-negative intraerythrocytic bacterium and the etiological agent of bovine Anaplasmosis. Even though considerable research efforts have been undertaken, Anaplasmosis vaccine development remains a challenging field. Outer-membrane-specific antigens responsible for the ability of more complex immunogens could have a significant role in the protective response. Thus, the identification of outer-membrane antigens represents a major goal in the development of bacterial vaccines. Considering that 40 % of the annotated proteins in A. marginale remain as hypothetical, we selected three candidate antigens, AM1108, AM127, and AM216 based on experimental evidence, in silico structure prediction of ß-barrel outer membrane, and orthology clustering. Sequence alignment and analysis demonstrated a high degree of conservation for the three proteins between the isolates from Argentina compared to the American strains. We confirmed the transcription of the three genes in the intraerythrocytic stage. AM1108 and AM216 recombinant proteins elicited specific T-cell response proliferation and a significant rise in TNF-α and IFN-γ transcript levels, respectively. Only AM1108 was able to be recognized by specific antibodies from infected bovines. This study allowed the identification of new candidate components of the outer-membrane fraction of A. marginale. Further studies will be required to analyze their potential as effective antigens for being included in rational vaccine strategies.

Multi-locus typing scheme for Babesia bovis and Babesia bigemina reveals high levels of genetic variability in strains from Northern Argentina.

Bovine babesiosis, caused by the protozoa Babesia bovis and Babesia bigemina, is a tick-borne disease distributed in tropical regions worldwide. Current control measures are based on the use of acaricides and live attenuated vaccines. The major economic impact of babesiosis lies in the cattle industry. In order to gain insight into the extent of genetic diversity in populations of parasites in the field, we developed two MLST schemes for the molecular genotyping of B. bigemina and B. bovis. We have also developed a custom-designed bioinformatic pipeline to facilitate the automated processing of raw sequences and further diversity and phylogenetic analysis. The overall MLST scheme exhibited the maximum discriminatory power (Simpson Index=1) for B. bovis and a high level of discrimination for B. bigemina (Simpson Index=0.9545). Genetic diversity was very high and infections with multiple genotypes were frequently found for both parasites in outbreak samples from the Northeast and Northwest of Argentina. Recombination events, which could have arisen from these multiple infections, were suggested by intra-loci linkage disequilibrium analysis and the lack of congruence in phylogenetic trees from individual genes. The two MLST schemes developed here are a robust, objective and easily adoptable technology to analyze the genetic diversity and population structure of parasites of the genus Babesia.

Phospholipase A1: a novel virulence factor in Trypanosoma cruzi.

Phospholipase A1 (PLA1) has been described in the infective stages of Trypanosoma cruzi as a membrane-bound/secreted enzyme that significantly modified host cell lipid profile with generation of second lipid messengers and concomitant activation of protein kinase C. In the present work we determined higher levels of PLA1 expression in the infective amastigotes and trypomastigotes than in the non-infective epimastigotes of lethal RA strain. In addition, we found similar expression patterns but distinct PLA1 activity levels in bloodstream trypomastigotes from Cvd and RA (lethal) and K98 (non-lethal) T. cruzi strains, obtained at their corresponding parasitemia peaks. This fact was likely due to the presence of different levels of anti-T. cruzi PLA1 antibodies in sera of infected mice, that modulated the enzyme activity. Moreover, these antibodies significantly reduced in vitro parasite invasion indicating the participation of T. cruzi PLA1 in the early events of parasite-host cell interaction. We also demonstrated the presence of lysophospholipase activity in live infective stages that could account for self-protection against the toxic lysophospholipids generated by T. cruzi PLA1 action. At the genome level, we identified at least eight putative genes that codify for T. cruzi PLA1 with high amino acid sequence variability in their amino and carboxy-terminal regions; a putative PLA1 selected gene was cloned and expressed as a recombinant protein that possessed PLA1 activity. Collectively, the results presented here point out at T. cruzi PLA1 as a novel virulence factor implicated in parasite invasion.