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Neospora caninum is a protozoan parasite that causes abortion and important economic losses in cattle worldwide. There are no treatments or vaccines available; disease control is based on diagnosis and herd management strategies. We developed, validated, and evaluated under field conditions a competitive inhibition ELISA based on the truncated SAG1 protein (tSAG1), expressed in Escherichia coli, and the RafNeo5 monoclonal antibody (ciELISAtSAG1). A criterion based on the 3-y sequential serologic analysis of 230 dairy cows by IFAT was used as the gold standard. The assay was validated using 860 serum samples from cows that were consistently positive or negative by IFAT throughout the study period. ciELISAtSAG1 was then used to evaluate the prevalence of neosporosis in 16 beef cow herds (22 samples per herd, 352 total samples). The results were compared with those from IFAT and a commercial cELISA (cELISAVMRD). The ciELISAtSAG1 cutoff was ≥ 29%I, with a diagnostic sensitivity of 98.7% (95% CI = 96.8–99.7%) and a diagnostic specificity of 97.9% (95% CI = 96.4–99.0%). Concordance among IFAT, cELISAVMRD, and ciELISAtSAG1 was 90.3%. The agreement (κ) between ciELISAtSAG1 and the other 2 tests was ≥ 0.81. The overall prevalence of neosporosis in the 16 beef herds was 30% (range: 5–60%). The ciELISAtSAG1 could be useful for large-scale detection of anti–N. caninum antibodies in cattle and seroepidemiologic investigations, given its appropriate sensitivity and specificity, and the simplicity of production. EEA Rafaela Fil: Novoa, María Belen. Instituto Nacional de Tecnología Agropecuaria (INTA). Estación Experimental Agropecuaria Rafaela; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina Fil: Valentini, Beatriz. Instituto Nacional de Tecnología Agropecuaria (INTA). Estación Experimental Agropecuaria Rafaela; Argentina Fil: Sarli, Macarena. Instituto Nacional de Tecnología Agropecuaria (INTA). Estación Experimental Agropecuaria Rafaela; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina Fil: Torioni, Susana. Instituto Nacional de Tecnología Agropecuaria (INTA). Estación Experimental Agropecuaria Rafaela; Argentina Fil: Primo, María Evangelina. Instituto Nacional de Tecnología Agropecuaria (INTA). Estación Experimental Agropecuaria Rafaela; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina Fil: Echaide, Ignacio. Instituto Nacional de Tecnología Agropecuaria (INTA). Estación Experimental Agropecuaria Rafaela; Argentina

Background Tritrichomonas foetus is the etiologic agent of the sexually transmitted disease Bovine Trichomonosis (BT).In Argentina, BT is endemic and represents a relevant health problem that causes reproductive inefficiency in cattle andlarge economic losses. Metronidazole is the drug of choice in the treatment of BT. Treatment has been associated with atemporary resolution of the clinical signs but is not able to control the disease. In recent years, the apparition of in vivo andin vitro aerobic and anaerobic resistance leading to ineffective treatments has been reported.Aims Thus, the aim of the present study was to explore the susceptibility of six different isolates of T. foetus under aerobic(AC) and anaerobic (ANC) conditions.Results and discussion Six isolates of T. foetus were obtained from samples of preputial smegma of bovine origin. Values ofminimum lethal concentration and minimum inhibitory concentration were higher than those observed in other works andrepresent current data in Argentina and provide information to establish new treatment protocols. Fil: Rivero, María Belén. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet Noa Sur. Instituto Multidisciplinario de Salud, Tecnología y Desarrollo. - Universidad Nacional de Santiago del Estero. Instituto Multidisciplinario de Salud, Tecnología y Desarrollo; Argentina. Universidad Nacional de Santiago del Estero. Facultad de Ciencias Médicas; Argentina Fil: Luque, Melchor Emilio. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet Noa Sur. Instituto Multidisciplinario de Salud, Tecnología y Desarrollo. - Universidad Nacional de Santiago del Estero. Instituto Multidisciplinario de Salud, Tecnología y Desarrollo; Argentina. Universidad Nacional de Santiago del Estero. Facultad de Ciencias Médicas; Argentina. Universidad Nacional de Santiago del Estero. Facultad de Agronomía y Agroindustrias; Argentina Fil: Abdala, María Eugenia. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet Noa Sur. Instituto Multidisciplinario de Salud, Tecnología y Desarrollo. - Universidad Nacional de Santiago del Estero. Instituto Multidisciplinario de Salud, Tecnología y Desarrollo; Argentina. Universidad Nacional de Santiago del Estero. Facultad de Ciencias Médicas; Argentina. Universidad Nacional de Santiago del Estero. Facultad de Agronomía y Agroindustrias; Argentina Fil: Luna, Bruno Elias. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet Noa Sur. Instituto Multidisciplinario de Salud, Tecnología y Desarrollo. - Universidad Nacional de Santiago del Estero. Instituto Multidisciplinario de Salud, Tecnología y Desarrollo; Argentina Fil: Di Lullo, David. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet Noa Sur. Instituto Multidisciplinario de Salud, Tecnología y Desarrollo. - Universidad Nacional de Santiago del Estero. Instituto Multidisciplinario de Salud, Tecnología y Desarrollo; Argentina Fil: Echaide, Ignacio Eduardo. Instituto Nacional de Tecnología Agropecuaria. Centro Regional Santa Fe. Estación Experimental Agropecuaria Rafaela; Argentina Fil: Carranza, Pedro Gabriel. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet Noa Sur. Instituto Multidisciplinario de Salud, Tecnología y Desarrollo. - Universidad Nacional de Santiago del Estero. Instituto Multidisciplinario de Salud, Tecnología y Desarrollo; Argentina. Universidad Nacional de Santiago del Estero. Facultad de Ciencias Médicas; Argentina. Universidad Nacional de Santiago del Estero. Facultad de Agronomía y Agroindustrias; Argentina Fil: Rivero, Fernando David. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet Noa Sur. Instituto Multidisciplinario de Salud, Tecnología y Desarrollo. - Universidad Nacional de Santiago del Estero. Instituto Multidisciplinario de Salud, Tecnología y Desarrollo; Argentina. Universidad Nacional de Santiago del Estero. Facultad de Ciencias Médicas; Argentina. Universidad Nacional de Santiago del Estero. Facultad de Agronomía y Agroindustrias; Argentina

La neosporosis es una enfermedad de gran impacto en el ganado vacuno debido a que causa abortos en el segundo y último trimestre de la gestación. En esta revisión se resume y discute la información sobre la misma, recopilada de dos décadas y media de estudios en bovinos de Argentina. Se aportan datos sobre el diagnóstico, la prevalencia y los avances en el estudio de la enfermedad. Está dirigida a los médicos veterinarios dedicados al diagnóstico y a la investigación de la neosporosis y/o a la producción bovina., Neosporosis is a disease with great impact in cattle, responsible for abortions in the second and last trimester of gestation. The present revision summarizes and discusses information from two and a half decades of studies about the disease in cattle from Argentina, contributing data about prevalence, diagnosis and progress in the knowledge of the disease. this report will be of interest for veterinarians specialized in diagnosis and investigation of neosporosis and/or bovine production., Facultad de Ciencias Veterinarias

The expansion of anaplasmosis to non-endemic areas in Argentina has created the need for specific treatments to eliminate Anaplasma marginale from carriers. The most recent studies have failed to chemosterilize A. marginale infections. In this work, we compare the efficacy of long-acting oxytetracycline (OTC) and imidocarb dipropionate (IMD) to chemosterilize the A. marginale infection. For this purpose, twenty steers were randomly clustered into two groups of ten animals each 78 days after A. marginale experimental infection (day 0). Cattle from group 1 (G1) were treated with three doses of 20 mg kg−1 of OTC (Terramycin® LA, 200 mg/ml) 7 days apart by intramuscular injection. Cattle from G2 were treated with two doses of 5 mg kg−1 of IMD (Imizol®, 120 mg/ml) 14 days apart by intramuscular injection. The efficacy of sterilizing treatments was evaluated by detection of DNA by nested PCR, anti-MSP5 antibodies by ELISA and by inoculation of splenectomized calves with blood from the steers 104 days post-treatment (dpt). The results showed 50% efficacy of the OTC treatment to chemosterilize persistent A. marginale infections in cattle and the failure of the IMD treatment under the evaluated conditions. The persistence of specific antibody levels in the sterilized animals (56 dpt) was shorter than the period of DNA detection. The ELISA was the test of choice to confirm the sterilizing outcome after 60 dpt. In spite of its limitations, the sterilization of A. marginale carrier status using OTC, could be useful for high-value bovines in non-endemic areas.

Anaplasma marginale is the most prevalent tick-borne livestock pathogen with worldwide distribution. Bovine anaplasmosis is a significant threat to cattle industry. Anaplasmosis outbreaks in endemic areas are prevented via vaccination with live A. centrale produced in splenectomized calves. Since A. centrale live vaccine can carry other pathogens and cause disease in adult cattle, research efforts are directed to develop safe recombinant subunit vaccines. Previous work found that the subdominant proteins of A. marginale type IV secretion system (T4SS) and the subdominant elongation factor-Tu (Ef-Tu) were involved in the protective immunity against the experimental challenge in cattle immunized with the A. marginale outer membrane (OM). This study evaluated the immunogenicity and protection conferred by recombinant VirB9.1, VirB9.2, VirB10, VirB11, and Ef-Tu proteins cloned and expressed in E. coli. Twenty steers were randomly clustered into four groups (G) of five animals each. Cattle from G1 and G2 were immunized with a mixture of 50 μg of each recombinant protein with Quil A® or Montanide™ adjuvants, respectively. Cattle from G3 and G4 (controls) were immunized with Quil A and Montanide adjuvants, respectively. Cattle received four immunizations at three-week intervals and were challenged with 107 A. marginale-parasitized erythrocytes 42 days after the fourth immunization. After challenge, all cattle showed clinical signs, with a significant drop of packed cell volume and a significant increase of parasitized erythrocytes (p

The aim of the present study was to characterize the specific immune response in prepubertal female calves inoculated with Neospora caninum. Forty-eight N. caninum-seronegative 6-month-old Angus female calves were randomly allocated into two groups: group A calves were inoculated subcutaneously (sc) with 1 × 106 tachyzoites of the low virulence NC-Argentina LP1 isolate in sterile phosphate-buffered saline (PBS); group B calves were mock inoculated sc with sterile PBS. Calves from group A developed a specific immune response characterized by the production of IgG antibodies and the expression of IFN-γ and TNF-α cytokines. Animals did not present any febrile reaction or reactions at the site of inoculation. Although chronic N. caninum infection was developed in 50% of calves of group A after inoculation, according to the presence of antibodies against rNc-SAG4, antigen characteristic of bradyzoites, N. caninum antibodies dropped below the cut-off of ELISA from day 210 post-inoculation onwards. Future trials using the same group of inoculated animals will allow the characterization of the evolution of the immune response during pregnancy and to determine whether the immunization with the local isolate is able to prevent congenital transmission and to protect against heterologous challenges., Facultad de Ciencias Veterinarias

N-glycosylation has remained mostly unexplored in Piroplasmida, an order of tick-transmitted pathogens of veterinary and medical relevance. Analysis of 11 piroplasmid genomes revealed three distinct scenarios regarding N-glycosylation: Babesia sensu stricto (s.s.) species add one or two N-acetylglucosamine (NAcGlc) molecules to proteins, Theileria equi and Cytauxzoon felis add (NAcGlc)2-mannose, while B. microti and Theileria s.s. synthesize dolichol-P-P-NAcGlc and dolichol-P-P-(NAcGlc)2 without subsequent transfer to proteins. All piroplasmids possess the gene complement needed for the synthesis of the N-glycosylation substrates, dolichol-P and sugar nucleotides. The oligosaccharyl transferase of Babesia species, T. equi and C. felis, is predicted to be composed of only two subunits, STT3 and Ost1. Occurrence of short N-glycans in B. bovis merozoites was experimentally demonstrated by fluorescence microscopy using a NAcGlc-specific lectin. In vitro growth of B. bovis was significantly impaired by tunicamycin, an inhibitor of N-glycosylation, indicating a relevant role for N-glycosylation in this pathogen. Finally, genes coding for N-glycosylation enzymes and substrate biosynthesis are transcribed in B. bovis blood and tick stages, suggesting that this pathway is biologically relevant throughout the parasite life cycle. Elucidation of the role/s exerted by N-glycans will increase our understanding of these successful parasites, for which improved control measures are needed.

Surface proteins bound to the cell membrane by glycosylphosphatidylinositol (GPI) anchors are considered essential for the survival of pathogenic protozoans. In the case of the tick-transmitted hemoparasite Babesia bovis, the most virulent causative agent of bovine babesiosis, the GPI-anchored proteome was recently unraveled by an in silico approach. In this work, one of the identified proteins, GASA-1 (GPI-Anchored Surface Antigen-1), was thoroughly characterized. GASA-1 is 179 aa long and has the characteristic features of a GPI-anchored protein, including a signal peptide, a hydrophilic core and a hydrophobic tail that harbors a GPI anchor signal. Transcriptomic analysis shows that it is expressed in pathogenic and attenuated B. bovis strains. Notably, the gasa-1 gene has syntenic counterparts in B. bigemina and B. ovata, which also encode GPI-anchored proteins. This is highly unusual since all piroplasmid GPI-anchored proteins described so far have been found to be species-specific. Sequencing of gasa-1 alleles from B. bovis geographical isolates originating from Argentina, USA, Brazil, Mexico and Australia showed over 98 % identity in both nucleotide and amino acid sequences. A recombinant form of GASA-1 (rGASA-1) was generated in E. coli and anti-rGASA-1 antibodies were raised in mice. Fixed and live immunofluorescence assays showed that GASA-1 is expressed in in vitro cultured B. bovis merozoites and surface-exposed. Moreover, incubation of B. bovis in vitro cultures with anti-GASA-1 antibodies partially, but significantly, reduced erythrocyte invasion, indicating that this protein bears neutralization-sensitive antibody epitopes. Splenocytes of rGASA-1-inoculated mice showed a specific proliferative response when exposed to the recombinant protein, indicating that GASA-1 bears T-cell epitopes. Finally, sera from a group of B. bovis-infected cattle reacted with the recombinant protein, demonstrating that GASA-1 is expressed during natural infection of bovines with B. bovis, and suggesting that it is immunodominant. The high degree of conservation among B. bovis isolates and the presence of syntenic genes in other Babesia species suggest a relevant role of GASA-1 and GASA-1-like proteins for parasite survival, especially considering that, due to their surface location, they are exposed to the selection pressure of the host immune system. The highlighted features of GASA-1 make it an interesting candidate for the development of vaccines against bovine babesiosis.

Water buffaloes ( Bubalus bubalis ) are raised in tropical and subtropical regions of the world, and act as hosts of Babesia bovis parasites and the tick vector Rhipicephalus microplus . As no clinical cases of B. bovis- infection have been reported, we hypothesized that, unlike bovines, water buffaloes respond asymptomatically to an acute infection. To test this hypothesis, we inoculated two groups of 24-month-old Mediterranean breed water buffaloes with 10 8 erythrocytes infected with two Argentine B. bovis isolates: BboM2P (n = 5) or BboS2P (n = 5). These strains displayed mild (BboM2P) or high (BboS2P) pathogenicity in Bos taurus calves of the same age (n = 5 and n = 1, respectively), when tested in parallel. In water buffaloes, no changes in body temperature were observed with both strains, and no hematocrit changes were detected in BboM2P-inoculated animals. In contrast, in the BboS2P-inoculated water buffalo group significant but relatively minor reductions in haematocrit values were noted compared to the infected bovine. The parasitemia attained in water buffaloes was considerably lower than in bovines and could only be detected by nested PCR, or indirectly via serology, whereas in most bovines, it could also be detected in Giemsa-stained smears under the light microscope. Our results show that water buffaloes present no or significantly mitigated clinical symptoms to B. bovis infections and suggest that they are able to substantially reduce and/or eliminate B. bovis parasites from circulation by an efficient innate immune mechanism.

We carried out an inter-laboratory trial to compare the serological tests commonly used for the detection of specific Neospora caninum antibodies in cattle in Ibero- American countries. A total of eight laboratories participated from the following countries: Argentina (n = 4), Brazil (n = 1), Peru (n = 1), Mexico (n = 1), and Spain (n = 1). A blind panel of well-characterized cattle sera (n = 143) and sera representative of the target population (n = 351) was tested by seven in-house indirect fluorescent antibody tests (IFATs 1–7) and three enzyme-linked immunosorbent assays (ELISAs 1–3; two in-house and one commercial). Diagnostic performance of the serological tests was calculated and compared according to the following criteria: (1) the BPre-test information,^ which uses previous epidemiological and serological data; (2) the BMajority of tests,^ which classifies a serumas positive or negative according to the results obtained by most tests evaluated. Unexpectedly, six tests showed either sensitivity (Se) or specificity (Sp) values lower than 90%. In contrast, the best tests in terms of Se, Sp, and area under the ROC curve (AUC) values were IFAT 1 and optimized ELISA 1 and ELISA 2. We evaluated a high number of IFATs, which are the most widely used tests in Ibero-America. The significant discordances observed among the tests regardless of the criteria employed hinder control programs and urge the use of a common test or with similar performances to either the optimized IFAT 1 and ELISAs 1 and 2., Facultad de Ciencias Veterinarias

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.

Infections by Babesia bovis limit cattle production and cause important economic losses in tropical and subtropical areas around the world. Monitoring of calf sera can be used to detect unprotected cattle herds and to decide on strategic control measures, as well as for epidemiological studies. Merozoite surface antigen 2c (MSA-2c) is an immunodominant surface protein expressed in B. bovis merozoites and sporozoites and contains B-cell epitopes that are conserved among geographic isolates. A monoclonal antibody against recombinant MSA-2c (rMSA-2c) was previously shown to inhibit the binding of anti- B. bovis antibodies to a parasite B-cell epitope in a competitive enzyme-linked immunosorbent assay (cELISA) format. In the work at hand, the parameters of this cELISA were reevaluated and adjusted when necessary, and a cutoff value was determined by receiver operator characteristic (ROC) curve analysis of a total of 357 bovine sera of known reactivity, as assessed by indirect immunofluorescence assay (IFAT). The established rMSA-2c cELISA demonstrated a specificity of 98% and a sensitivity of 96.2%. An additional set of 303 field bovine sera from regions where ticks are endemic and tick-free regions of Argentina was tested by both rMSA-2c cELISA and IFAT, and the results were shown to be in very good agreement (kappa index, 0.8325). The performance shown by rMSA-2c cELISA in the detection of B. bovis -specific antibodies and its suitability for standardization and large-scale production, as well as the possibility of its application in most veterinary diagnostic laboratories, make the assay a powerful tool for the surveillance of herd immunity as a strategic measure for the control of bovine babesiosis.

The aim of this study was to characterize the pathogenesis of Neospora caninum in experimentally inoculated pregnant water buffalo (Bubalus bubalis). Twelve Mediterranean female water buffaloes ranging in age from 4 to 14 years old and seronegative to N. caninum by indirect fluorescent antibody test (IFAT) were involved. Ten females were intravenously inoculated with 10(8) tachyzoites of NC-1 strain at 70 (n=3) or 90 (n=7) days of pregnancy (dp). Two control animals were inoculated with placebo at 70 and 90 dp, respectively. Serum samples were obtained weekly following inoculation to the end of the experiment. Three animals inoculated at 70 dp were slaughtered at 28 days post inoculation (dpi), three animals inoculated at 90 dp were slaughtered at 28 dpi and the remaining four animals inoculated at 90 dp were slaughtered at 42 dpi. Fetal fluids from cavities and tissue samples were recovered for IFAT and histopathology, immunohistochemistry and PCR, respectively. Genomic DNA from fetal tissues was used for parasite DNA detection and microsatellite genotyping in order to confirm the NC-1 specific-infection. Dams developed specific antibodies one week after the inoculation and serological titers did not decrease significantly to the end of the experiment. No abortions were recorded during the experimental time; however, one fetus from a dam inoculated at 70 dp was not viable at necropsy. Specific antibodies were detected in only two fetuses from dams inoculated at 90 dp that were slaughtered at 42 dpi. No macroscopic changes in the placentas and organs of viable fetuses were observed. Nonsuppurative placentitis was a common microscopic observation in Neospora-inoculated specimens. Microscopic fetal lesions included nonsuppurative peribronchiolar interstitial pneumonia, epicarditis and myocarditis, interstitial nephritis, myositis and periportal hepatitis. Positive IHC results were obtained in two fetuses from dams inoculated at 70 dp and slaughtered at 28 dpi. N. caninum DNA was detected in placentas and fetuses from all inoculated animals. The pattern of amplified microsatellites from placental and fetal tissues resembled the NC-1 strain. Water buffaloes, like cattle, are susceptible to experimental inoculation with N. caninum at early pregnancy.

Completion of the Babesia bovis (T2Bo strain) genome provides detailed data concerning the predicted proteome of this parasite, and allows for a bioinformatics approach to gene discovery. Comparative genomics of the hemoprotozoan parasites B. bovis and Theileria parva revealed a highly conserved syntenic block of genes flanking the p67 gene of T. parva, a sporozoite stage-specific vaccine candidate against East Coast fever (ECF). The syntenic gene in B. bovis, designated bov57, encodes a protein of limited amino acid sequence identity (11.8%) to p67. Monoclonal antibodies were produced against recombinant BOV57 and were used to demonstrate expression of BOV57 in merozoite and kinete stages of the T2Bo strain of B. bovis. Transcript levels of bov57 in kinetes were increased 100-fold in comparison to msa-1, a previously identified gene encoding an erythrocyte stage surface protein. Amino acid sequence comparisons between the T2Bo strain and two attenuated and virulent strains from Argentina and Australia revealed a high degree of sequence conservation in BOV57 among these geographically and pathogenically divergent isolates (97% amino acid sequence identity). Additional genomic comparisons show that the bov57 gene locus is also conserved in Babesia bigemina and Babesia equi. While not identifiable through amino acid or nucleotide sequence similarity, the conserved gene order within this locus in multiple piroplasms may suggest a critical function adapted for each species' unique host and life-cycle.

Babesia bovis is a tick-borne apicomplexan pathogen that remains an important constraint for the development of cattle industries worldwide. The existence of different strains and subpopulations has long been described in this hemoparasite. However, few molecular markers have been reported for strain genotyping and characterization. Minisatellite sequences show high levels of variation and therefore provide excellent tools for both the genotyping and population genetic analysis. In this work we report a set of five molecular markers containing minisatellites that showed a variable degree of polymorphism in several American strains. We have used a bioinformatics approach to search for marker sequences contained in open reading frames. Five genes were chosen and primers were designed in conserved regions flanking the repeat region. Two of the genes were the previously described Bv80/Bb-1 and TRAP. The other three genes were named p200, Antigen 3 and Desmoyokin. Amplification by PCR, sequencing and comparative analysis of 11 strains from Argentina, Brazil, Uruguay, Mexico and USA determined that the tandem repeats varied in number and sequence among the isolates. Genome analysis of the five markers revealed that they were single copy and distributed across the four B. bovis chromosomes. When the new markers were analyzed in an experimental infection, absolute sequence conservation was found, indicating the stability of these markers during the course of infection. These markers were also stable during three syringe passages through calves. The application of this panel of molecular markers could provide new molecular tools for the genotyping of B. bovis isolates and analysis of changes in parasite populations following vaccination.

The merozoite surface antigen-2 (msa-2) family of Babesia bovis is a group of variable genes that share conserved 5' and 3' ends and encode for membrane-anchored glycoproteins that have been postulated as vaccine candidates. In this work, we analyzed the sequences of three of these genes (msa-2a1, a2, and 2b) from two geographically distant strains and detected a certain degree of genotypic diversity that could be exploited to work out new molecular tools for the discrimination of B. bovis field samples. Here we describe a PCR restriction assay that was developed based on this observation and tested on several B. bovis strains and isolates. The results show a strain-specific band pattern in geographically distant isolates, indicating the presence of differentially located BspMI restriction sites. This approach provides a simple method for the differentiation of American B. bovis strains.

Bovine babesiosis caused by Babesia bovis is a disease that hampers the production of beef and dairy cattle in tropical and subtropical regions of the world. New diagnostic methods based on recombinant antigens constitute valuable biotechnological tools for the strategic control of this disease. We have developed a competitive enzyme-linked immunosorbent assay that includes a recombinant form of the merozoite surface antigen-2c and a novel monoclonal antibody against it. Preliminary results showed that this test is able to identify specific antibodies against B. bovis from experimentally and naturally infected cattle.

Graphical abstract Bovipain-2 (in red), a Babesia bovis cysteine protease, is released into the host erythrocyte. Bovipain-2 is expressed in both virulent and attenuated B. bovis. DAPI stains the nuclei of two B. bovis. Highlights ► Genome sequence of the virulent T2Bo strain reveals 66 peptidases which constitute 2% of the annotated coding sequences. ► Each of the peptidases identified in the virulent parent was represented in the attenuated daughter strain. ► Microarrays analyses of transcript levels for all 66 peptidase-encoding genes were investigated. ► Our study discusses our findings in regards to peptidase involvement in in vivo attenuation of B. bovis., Identifying virulence determinants in Apicomplexan parasites remains a major gap in knowledge for members within this phylum. We hypothesized that peptidases would segregate with virulence between a virulent parent Babesia bovis strain and an attenuated daughter strain derived by rapid in vivo passage. Using the complete genome sequence of the virulent T2Bo strain, 66 peptidases were identified and active sites confirmed. The presence, sequence identity and expression levels were tested for each of the 66 peptidases in the virulent parent and attenuated daughter T2Bo strains using whole genome, targeted sequencing approaches and microarrays analyses. Quantitative PCR revealed that there was no significant difference in peptidase expression between the virulent and attenuated strains. We conclude that while peptidases may well play a required role in B. bovis pathogenesis, neither loss of peptidase gene content nor reduced gene expression underlies the loss of virulence associated with in vivo passage and attenuation.

The population structure of original Babesia bigemina isolates and reference strains with a defined phenotypic profile was assessed using 18S rRNA and rap-1c genes. Two reference strains, BbiS2P-c (virulent) and BbiS1A-c (low virulence), were biologically cloned in vitro. The virulence profile of the strains and clones was assessed in vivo. One fully virulent and one low-virulence clone were mixed in identical proportions to evaluate their growth efficiency in vitro. Each clone was differentiated by two microsatellites and the gene gp45. The 18S rRNA and rap-1c genes sequences from B. bigemina biological clones and their parental strains, multiplied exclusively in vivo or in vitro, were compared with strain JG-29. The virulence of clones derived from the BbiS2P-c strain was variable. Virulent clone Bbi9P1 grew more efficiently in vitro than did the low-virulence clone Bbi2A1. The haplotypes generated by the nucleotide polymorphism, localized in the V4 region of the 18S rRNA, allowed the identification of three genotypes. The rap-1c haplotypes allowed defining four genotypes. Parental and original strains were defined by multiple haplotypes identified in both genes. The rap-1c gene, analyzed by high-resolution melting (HRM), allowed discrimination between two genotypes according to their phenotype, and both were different from JG-29. B. bigemina biological clones made it possible to define the population structure of isolates and strains. The polymorphic regions of the 18S rRNA and rap-1c genes allowed the identification of different subpopulations within original B. bigemina isolates by the definition of several haplotypes and the differentiation of fully virulent from low virulence clones. Fil: Thompson, Carolina Soledad. Instituto Nacional de Tecnología Agropecuaria. Centro Regional Santa Fe. Estación Experimental Agropecuaria Rafaela; Argentina Fil: Baravalle, María Eugenia. Instituto Nacional de Tecnología Agropecuaria. Centro Regional Santa Fe. Estación Experimental Agropecuaria Rafaela; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina Fil: Valentini, B.. Instituto Nacional de Tecnología Agropecuaria. Centro Regional Santa Fe. Estación Experimental Agropecuaria Rafaela; Argentina Fil: Mangold, Atilio Jose. Instituto Nacional de Tecnología Agropecuaria. Centro Regional Santa Fe. Estación Experimental Agropecuaria Rafaela; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina Fil: Torioni de Echaide, S.. Instituto Nacional de Tecnología Agropecuaria. Centro Regional Santa Fe. Estación Experimental Agropecuaria Rafaela; Argentina Fil: Ruybal, Paula. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Investigaciones en Microbiología y Parasitología Médica. Universidad de Buenos Aires. Facultad de Medicina. Instituto de Investigaciones en Microbiología y Parasitología Médica; Argentina. Instituto Nacional de Tecnología Agropecuaria. Centro de Investigaciones Agropecuarias; Argentina Fil: Farber, Marisa Diana. Instituto Nacional de Tecnología Agropecuaria. Centro de Investigación en Ciencias Veterinarias y Agronómicas. Instituto de Biotecnología; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina Fil: Echaide, Ignacio Eduardo. Instituto Nacional de Tecnología Agropecuaria. Centro Regional Santa Fe. Estación Experimental Agropecuaria Rafaela; Argentina

Glycosylphosphatidylinositol-anchored proteins are abundant on the surface of pathogenic protozoans and might play an important role for parasite survival. In the present work, the relevance of GPI-anchored proteins for erythrocyte invasion of the cattle hemoparasite Babesia bovis was studied. We show that cleavage of GPI-anchored antigens from the merozoite parasite stage by phosphatidylinositol-specific phospholipase C abolished invasion of erythrocytes demonstrating the importance of this class of molecules for parasite propagation. In addition, the repertoire of GPI-anchored proteins of B. bovis was predicted with high fidelity by searching its genome with available web-based bioinformatic tools. Altogether 17 GPI-anchored proteins were identified, 5 of which represent the already characterized variable merozoite surface antigens (VMSAs). Fifteen of the identified GPI-anchored proteins contain 2–26 amino acid repeats indicating that they are likely involved in functions of recognition, adhesion, or transport. Repeats were found to contain an increased frequency of proline, indicative of unstructured regions; and were estimated to be 3.21 times more hydrophilic than non-repeat regions. This suggests that they might represent eminent antibody epitopes. The majority of the putative GPI-anchored antigens reported in this work have so far remained unnoticed, though they may represent potential candidates for inclusion in a subunit vaccine. Fil: Rodriguez, Anabel Elisa. Instituto Nacional de Tecnología Agropecuaria. Centro de Investigación en Ciencias Veterinarias y Agronómicas. Instituto de Patobiología; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina Fil: Jacobsen, Monica Ofelia. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Instituto Nacional de Tecnología Agropecuaria. Centro de Investigación en Ciencias Veterinarias y Agronómicas. Instituto de Patobiología; Argentina Fil: Flores, Daniela Agustina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Instituto Nacional de Tecnología Agropecuaria. Centro de Investigación en Ciencias Veterinarias y Agronómicas. Instituto de Patobiología; Argentina. Ministerio de Ciencia. Tecnología e Innovación Productiva. Agencia Nacional de Promoción Cientifíca y Tecnológica; Argentina Fil: Echaide, Ignacio Eduardo. Instituto Nacional de Tecnología Agropecuaria. Centro Regional Santa Fe. Estación Experimental Agropecuaria Rafaela; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina Fil: Suarez, Carlos Esteban. Washington State University; Estados Unidos Fil: Schnittger, Leonhard. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Instituto Nacional de Tecnología Agropecuaria. Centro de Investigación en Ciencias Veterinarias y Agronómicas. Instituto de Patobiología; Argentina

Background: Loss of virulence is a phenotypic adaptation commonly seen in prokaryotic and eukaryotic pathogens. This mechanism is not well studied, especially in organisms with multiple host and life cycle stages such as Babesia, a tick-transmitted hemoparasite of humans and animals. B. bovis, which infects cattle, has naturally occurring virulent strains that can be reliably attenuated in vivo. Previous studies suggest the virulence loss mechanism may involve post-genomic modification. We investigated the transcriptome profiles of two geographically distinct B. bovis virulent and attenuated strain pairs to better understand virulence loss and to gain insight into pathogen adaptation strategies. Results: Expression microarray and RNA-sequencing approaches were employed to compare transcriptome profiles of two B. bovis strain pairs, with each pair consisting of a virulent parental and its attenuated derivative strain. Differentially regulated transcripts were identified within each strain pair. These included genes encoding for VESA1, SmORFs, undefined membrane and hypothetical proteins. The majority of individual specific gene transcripts differentially regulated within a strain were not shared between the two strains. There was a disproportionately greater number of ves genes upregulated in the virulent parental strains. When compared with their attenuated derivatives, divergently oriented ves genes were included among the upregulated ves genes in the virulent strains, while none of the upregulated ves genes in the attenuated derivatives were oriented head to head. One gene family whose specific members were consistently and significantly upregulated in expression in both attenuated strains was spherical body protein (SBP) 2 encoding gene where SBP2 truncated copies 7, 9 and 11 transcripts were all upregulated. Conclusions: We conclude that ves heterodimer pair upregulation and overall higher frequency of ves gene expressions in the virulent strains is consistent with the involvement of this gene family in virulence. This is logical given the role of VESA1 proteins in cytoadherence of infected cells to endothelial cells. However, upregulation of some ves genes in the attenuated derivatives suggests that the consequence of upregulation is gene-specific. Furthermore, upregulation of the spherical body protein 2 gene family may play a role in the attenuated phenotype. Exactly how these two gene families may contribute to the loss or gain of virulence is discussed. EEA Rafaela Fil: Pedroni, Monica J. Washington State University. College of Veterinary Medicine. Department of Veterinary Microbiology & Pathology. Program of Genomics; Estados Unidos Fil: Sondgeroth, Kerry S. Washington State University. College of Veterinary Medicine. Department of Veterinary Microbiology & Pathology. Program of Genomics; Estados Unidos Fil: Gallego-Lopez, Gina M. Washington State University. College of Veterinary Medicine. Department of Veterinary Microbiology & Pathology. Program of Genomics; Estados Unidos Fil: Echaide, Ignacio Eduardo. Instituto Nacional de Tecnología Agropecuaria (INTA). Estación Experimental Agropecuaria Rafaela. Laboratorio de Inmunología y Parasitología; Argentina Fil: Lau, Audrey OT. Washington State University. College of Veterinary Medicine. Department of Veterinary Microbiology & Pathology. Program of Genomics; Estados Unidos. Washington State University. College of Veterinary Medicine. Paul G. Allen School for Global Animal Health; Estados Unidos

Babesia bovis is a tick-transmitted haemoprotozoan and a causative agent of bovine babesiosis, a cattle disease that causes significant economic loss in tropical and subtropical regions. A panel of nineteen micro- and minisatellite markers was used to estimate population genetic parameters of eighteen parasite isolates originating from different continents, countries and geographic regions including North America (Mexico, USA), South America (Argentina, Brazil), the Middle East (Israel) and Australia. For eleven of the eighteen isolates, a unique haplotype was inferred suggesting selection of a single genotype by either in vitro cultivation or amplification in splenectomized calves. Furthermore, a high genetic diversity (H = 0.780) over all marker loci was estimated. Linkage disequilibrium was observed in the total study group but also in sample subgroups from the Americas, Brazil, and Israel and Australia. In contrast, corresponding to their more confined geographic origin, samples from Israel and Argentina were each found to be in equilibrium suggestive of random mating and frequent genetic exchange. The genetic differentiation (FST) of the total study group over all nineteen loci was estimated by analysis of variance (Θ) and Nei's estimation of heterozygosity (GST') as 0.296 and 0.312, respectively. Thus, about 30% of the genetic diversity of the parasite population is associated with genetic differences between parasite isolates sampled from the different geographic regions. The pairwise similarity of multilocus genotypes (MLGs) was assessed and a neighbour-joining dendrogram generated. MLGs were found to cluster according to the country/continent of origin of isolates, but did not distinguish the attenuated from the pathogenic parasite state. The distant geographic origin of the isolates studied allows an initial glimpse into the large extent of genetic diversity and differentiation of the B. bovis population on a global scale. Fil: Flores, Daniela Agustina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Instituto Nacional de Tecnología Agropecuaria. Centro Regional Buenos Aires; Argentina Fil: Minichiello, Y.. Instituto Nacional de Tecnología Agropecuaria. Centro Regional Buenos Aires; Argentina Fil: Araujo, F. R.. Ministerio da Agricultura Pecuaria e Abastecimento de Brasil. Empresa Brasileira de Pesquisa Agropecuaria; Brasil Fil: Shkap, V.. Kimron Veterinary Institute Israel; Israel Fil: Benítez, D.. Instituto Nacional de Tecnología Agropecuaria. Centro Regional Corrientes. Estación Experimental Agropecuaria Mercedes; Argentina Fil: Echaide, Ignacio Eduardo. Instituto Nacional de Tecnología Agropecuaria. Centro Regional Santa Fe. Estación Experimental Agropecuaria Rafaela; Argentina Fil: Rolls, P.. Tick Fever Centre; Australia Fil: Mosqueda, J.. Universidad Autónoma de Querétaro; México Fil: Pacheco, G. M.. Instituto Nacional de Tecnología Agropecuaria. Centro Regional Buenos Aires; Argentina Fil: Petterson, M.. Instituto Nacional de Tecnología Agropecuaria. Centro Regional Buenos Aires; Argentina Fil: Jacobsen, Monica Ofelia. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Instituto Nacional de Tecnología Agropecuaria. Centro Regional Buenos Aires; Argentina Fil: Schnittger, Leonhard. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Instituto Nacional de Tecnología Agropecuaria. Centro Regional Buenos Aires; Argentina

Background Virulence acquisition and loss is a dynamic adaptation of pathogens to thrive in changing milieus. We investigated the mechanisms of virulence loss at the whole genome level using Babesia bovis as a model apicomplexan in which genetically related attenuated parasites can be reliably derived from virulent parental strains in the natural host. We expected virulence loss to be accompanied by consistent changes at the gene level, and that such changes would be shared among attenuated parasites of diverse geographic and genetic background. Results Surprisingly, while single nucleotide polymorphisms in 14 genes distinguished all attenuated parasites from their virulent parental strains, all non-synonymous changes resulted in no deleterious amino acid modification that could consistently be associated with attenuation (or virulence) in this hemoparasite. Interestingly, however, attenuation significantly reduced the overall population's genome diversity with 81% of base pairs shared among attenuated strains, compared to only 60% of base pairs common among virulent parental parasites. There were significantly fewer genes that were unique to their geographical origins among the attenuated parasites, resulting in a simplified population structure among the attenuated strains. Conclusions This simplified structure includes reduced diversity of the variant erythrocyte surface 1 (ves) multigene family repertoire among attenuated parasites when compared to virulent parental strains, possibly suggesting that overall variance in large protein families such as Variant Erythrocyte Surface Antigens has a critical role in expression of the virulence phenotype. In addition, the results suggest that virulence (or attenuation) mechanisms may not be shared among all populations of parasites at the gene level, but instead may reflect expansion or contraction of the population structure in response to shifting milieus.

The virulence phenotype of Babesia bovis subpopulations was evaluated using biological clones derived from the high-virulence BboS2P and the low-virulence BboR1A strain and two original virulent isolates, BboL15 and BboL17, multiplied extensively in vitro or attenuated by successive passages in splenectomized calves. The virulence phenotype was assessed both by inoculation of normal Holstein adult steers and by analyses of polymorphic fragments of the single-copy Bv80 gene as a subpopulation marker. BboS2P and its nine derived clones contained a single 750 bp fragment with identical nucleotide sequences and numbers of repeats. A single fragment of approximately 850 bp was observed in BboR1A and its derived clones (Ca3B1, Ca2B1). Ca3B1 and Ca2B1 were differentiated by a stable deletion of 15 contiguous nucleotides in the Bv80 allele of Ca3B1. Both alleles were identified in the parental strain. Original isolates BboL15 and BboL17 contained two Bv80 fragments of different sizes. Interestingly, the heavy and light fragments persisted in the in vivo-attenuated strains and the virulent in vitro-multiplied strains, respectively. Despite the inter-strain allelic diversity of the Bv80 gene, the fragments had identical nucleotide sequences and numbers of repeats compared to their respective parental Bv80 genes. The high-virulence and low-virulence phenotypes remained unchanged after they were multiplied in vitro. In conclusion, the polymorphic B. bovis Bv80 gene, was a useful marker for differentiating subpopulations with different phenotypes. The brevity of the procedure to isolate one parasite from the original isolate or strain before in vitro cloning and the fact that the continuous in vitro multiplication did not modify the virulence phenotype of B. bovis clones strongly suggest that the in vivo-attenuated subpopulations existed in the original isolates before they were selected by passages in splenectomized calves.

The aim of this study was to compare the immune responses to live Neospora caninum tachyzoites and N. caninum native antigens formulated with immune stimulating complexes matrix (ISCOM-matrix) in calves. Fifteen calves were used in this study: 3 were intravenously inoculated with 1 × 10(8) live tachyzoites (Group A), 3 were inoculated twice with N. caninum native antigens formulated with ISCOMs (Group B); 3 with N. caninum native antigens in phosphate-buffered saline (PBS) (Group C); 3 received ISCOM-matrix (ISCOMs without antigen) (Group D) and 3 were negative controls receiving PBS (Group E). The last four groups were inoculated subcutaneously. The specific total IgG and its subtypes were analyzed by an indirect enzyme-linked immunosorbent assays (ELISAs) and by Western blot. IFN-γ levels in plasma was quantified using a commercial kit. All calves were challenged intravenously with 1 × 10(8) live tachyzoites at week 11 after receiving the first dose. Parasitemia was assessed in plasma samples by semi-nested PCR. Neospora-specific antibodies were detected in animals from Groups A and B in the week 2 after inoculation. The ELISA OD values were higher in Group B compared with Group A from weeks 6 to 11 (P

The merozoite surface antigens MSA-2 of Babesia bovis constitute a family of polymorphic GPI-anchored glycoproteins located at the parasite cell surface, that contain neutralization-sensitive B-cell epitopes. These are therefore putative vaccine candidates for bovine babesiosis. It was previously shown that (i) the MSA-2 antigens of the biologically cloned Mo7 strain are encoded by four tandemly organized genes: msa-2a(1), a(2), b and c, and (ii) at least one allele of each of these genes is present in the Argentine R1A strain with a moderate degree of polymorphism. The present work was aimed at defining neutralization-sensitive B-cell epitopes in the MSA-2 family, that are conserved among different B. bovis geographical isolates. To this end, msa-2a, b and c alleles from different isolates from Argentina, USA and Mexico were amplified by PCR, cloned and sequenced. Bioinformatic analysis by ClustalW alignments and B-cell epitope prediction algorithms performed on these sequences allowed the identification of several regions containing putative conserved B-cell epitopes. Four peptides representing these regions: (KDYKTMVKFCN from msa-2a(1); YYKKHIS, from msa-2b; and THDALKAVKQLIKT and ELLKLLIEA from msa-2c) were chemically synthesized, conjugated to keyhole limpet hemocyanin and used to inoculate mice to obtain immune sera. Anti-peptide antibodies recognized B. bovis merozoite extracts in all cases in ELISA tests. In addition, these sera reacted with the surface of merozoites of an Argentine and a Mexican B. bovis strains in immunofluorescence assays, and sera against two of the selected peptides inhibited invasion of erythrocytes by in vitro cultured merozoites. Taken together, the results show that the peptide sequences selected by bioinformatic analysis represent expressed and geographically conserved B. bovis B-cell epitopes that might be strong candidates for development of subunit vaccines.

Autonomous glycosylphosphatidylinositol (GPI) molecules (also protein-free GPIs or free GPIs) have been reported to be particularly abundant in some parasitic protozoa and mediate strong immunomodulatory effects on the host immune system. In the work at hand we have investigated the existence of free GPIs in Babesia bovis. Comparative thin layer chromatographic analysis of the protein-free glycolipid fraction of in vitro cultured B. bovis merozoites and erythrocyte membranes demonstrated the presence of an abundant parasite-specific band. Its chemical analysis revealed a GPI species containing a chain of two mannose residues, N-glucosamine and non-acylated inositol. The lipid moiety linked to inositol was diacylglycerol. The total fatty acid composition showed predominantly long-carbon chain molecules (12% of C(22:0) and 45% of C(24:0)). The potential of B. bovis to assemble the presented free GPI species was verified by the existence of seven genes in its genome that putatively encode the following GPI biosynthetic enzymes: PI N-acetyl-GlcN-transferase (PIG-A and GPI-1), N-acetyl-GlcN-PI-de-N-acetylase (PIG-L), acyltransferase (PIG-W), dolichyl-phosphate mannosyl transferase (DPM-1), GPI mannosyltransferase I (PIG-M), and GPI mannosyltransferase II (PIG-V). GPI biosynthesis is vital for the intraerythrocytic parasite stage as mannosamine, an inhibitor of GPI biosynthesis, impaired in vitro growth of B. bovis merozoites. Absence of the vast majority of N-glycan metabolism encoding genes in the B. bovis genome underscores that the growth inhibitory effect of mannosamine is attributable to its interference with GPI biosynthesis and not with assembly of N-linked oligosaccharides, as has been described for higher eukaryotes. Elucidation of the structure and biosynthesis of GPI may allow to facilitate the development of future immune interventions against bovine babesiosis.

Mini- and microsatellite sequences have proven to be excellent tools for the differentiation of strains and populations in several protozoan parasites due to their high variability. In the present work we have searched the genome of the tick-transmitted bovine hemoprotozoon Babesia bovis for tandem repeats (TRs) that could be useful for a multilocus typing system. Hundred and nineteen sequences were shortlisted and tested in five common B. bovis reference isolates originating from distinct geographic locations of North and South America: Texas, USA (T2Bo), Mexico (RAD and Mo7), and Santa Fe and Salta, Argentina (R1A and S2P, respectively). Satellite sequences were PCR-amplified using specific primers, separated by polyacrylamide gel electrophoresis, visualized by silver staining and sized. Fourteen TR sequences could be reliably amplified in all isolates and displayed length polymorphism. All primers used were specific for B. bovis and did not amplify genomic DNA from the bovine host or from Babesia bigemina, the principal co-infecting bovine parasite in the Americas, allowing their future use in field surveys. The 14 satellite markers identified are distributed throughout the four chromosomes of B. bovis as follows: chromosome 1 (n=3), chromosome 2 (n=2), chromosome 3 (n=5), and chromosome 4 (n=4). Within the five B. bovis isolates we identified nine satellite marker loci with two alleles, three with three alleles, one with four and another with five alleles. In comparison to Theileria parva, a bovine hemoprotozoan that pertains to the same piroplasmida order and own a genome of similar size, the number of polymorphic TRs and the average number of alleles per TR locus seem to be significantly reduced in the B. bovis genome. Furthermore, the ratio of micro- to minisatellites in both B. bovis and T. parva is considerably lower than in other eukaryotes, as confirmed by bioinformatic analysis. The multilocus genotype of the five B. bovis isolates was assessed and the genetic distance between each other determined followed by cluster analysis based on neighbor joining. The resulting phenogram showed that B. bovis isolates segregated into three clusters according to their geographic origin. The presented marker system is suitable to explore various parameters of B. bovis populations such as genetic diversity, infection dynamics and their structure under different epidemiological situations, which are of crucial importance for improved control strategies. Fil: Perez Llaneza, Agustina. Instituto Nacional de Tecnología Agropecuaria. Centro Nacional de Investigaciones Agropecuarias Castelar. Centro de Investigación en Ciencias Veterinarias y Agronómicas. Instituto de Patobiología; Argentina Fil: Caballero, Marina. Instituto Nacional de Tecnología Agropecuaria. Centro Nacional de Investigaciones Agropecuarias Castelar. Centro de Investigación en Ciencias Veterinarias y Agronómicas. Instituto de Patobiología; Argentina Fil: Baravalle, María Eugenia. Instituto Nacional de Tecnología Agropecuaria. Centro Regional Córdoba. Estación Exptal.reg.agrop.rafaela; Argentina Fil: Mesplet, Maria. Instituto Nacional de Tecnología Agropecuaria. Centro Nacional de Investigaciones Agropecuarias Castelar. Centro de Investigación en Ciencias Veterinarias y Agronómicas. Instituto de Patobiología; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina Fil: Mosqueda, Juan. Universidad Autónoma de Querétaro; México Fil: Suarez, Carlos Esteban. United States Department of Agriculture. Agricultural Research Service; Estados Unidos Fil: Echaide, Ignacio Eduardo. Instituto Nacional de Tecnología Agropecuaria. Centro Regional Córdoba. Estación Exptal.reg.agrop.rafaela; Argentina Fil: Katzer, Frank. The Moredun Research Institute; Reino Unido Fil: Pacheco, Maria Gabriela. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Instituto Nacional de Tecnología Agropecuaria. Centro de Investigación en Ciencias Veterinarias y Agronómicas. Instituto de Genética; Argentina Fil: Florin Christensen, Mónica. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Instituto Nacional de Tecnología Agropecuaria. Centro Nacional de Investigaciones Agropecuarias Castelar. Centro de Investigación en Ciencias Veterinarias y Agronómicas. Instituto de Patobiología; Argentina Fil: Schnittger, Leonhard. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Instituto Nacional de Tecnología Agropecuaria. Centro Nacional de Investigaciones Agropecuarias Castelar. Centro de Investigación en Ciencias Veterinarias y Agronómicas. Instituto de Patobiología; Argentina

Molecular detection of Babesia bigemina involves a nested PCR protocol and reverse line blot hybridization (RLBH) assay based on the 18S gene. In this study, we report the development of molecular tools for improving B. bigemina detection in bovine blood-a one-step PCR assay based on the amplification of rap-1a paralogous and a new RLBH Babesia spp. 18S probe. The one-step PCR assay is highly specific, with an estimated analytical sensitivity corresponding to 0.00002% parasitemia. The RLBH assay, with a new B. bigemina probe, allows the detection of all tested B. bigemina isolates showing no cross-hybridization with B. bovis 18S gene. By developing this highly specific and sensitive one-step PCR and upgrading the RLBH assay for B. bigemina, we have improved molecular assays which, together with serologic methods, provide valuable tools for epidemiologic studies of bovine babesiosis.

Bovine anaplasmosis caused by Anaplasma marginale is a worldwide major constraint to cattle production. The A. marginale major surface protein 1 alpha (msp1α) gene contains a variable number of tandem repeats in the amino terminal region and has been used for the characterization of pathogen genetic diversity. This study reports the first characterization of A. marginale genetic diversity in Argentina based on msp1α genotypes and its putative relationship with Rhipicephalus (Boophilus) microplus infestations. Herein, we analyzed whole blood bovine samples from anaplasmosis outbreaks in R. microplus infested (9 samples) and eradicated/free (14 samples) regions. Sequence analysis revealed the existence of 15 different msp1α genotypes with 31 different repeat units. Six new repeat sequences were discovered in this study and 13/31 (42%) repeats were unique to Argentinean strains. The analysis of msp1α repeat sequences according to R. microplus infestations resulted in three repeat groups: (i) found in tick-infested regions (20 repeats), (ii) found in tick free regions (6 repeats) and (iii) randomly distributed (5 repeats). Moreover, A. marginale msp1α genetic diversity was higher in tick-infested regions than in tick free areas. These results, together with previous evidence suggesting that A. marginale msp1α repeat units co-evolved with the tick vector, might represent an evidence of the role of tick-mediated transmission for the generation of pathogen genetic diversity., This work was supported by INCO Epigenevac project -UE FP6-, CONICET, ANPCyT and INTA, Argentina, Oklahoma Agricultural Experiment Station Project 1669.

Bovine babesiosis is caused by Babesia bovis and B. bigemina in Argentina. These protozoans are prevalent north of parallel 30 degrees S, where their natural vector Rhipicephalus (Boophilus) microplus is widespread. To prevent babesiosis outbreaks in endemic areas, an increasing population of 4-10-month-old calves are vaccinated with low virulence B. bovis R1A (BboR1A) and B. bigemina S1A (BbiS1A) strains. In non-endemic areas, an additional calf population is also vaccinated and boostered as adults, before they are relocated to R. microplus-endemic areas of the country. Serological tests are currently utilized not only to determine the status of natural Babesia spp. infections, but also to confirm the infection caused by vaccine strains. For this purpose, an indirect enzyme immunoassay (ELISA) based on the recombinant major surface antigen-2c (rMSA-2c) of B. bovis expressed in Escherichia coli, was standardized using sera from Babesia spp. experimentally infected cattle. ELISA(rMSA-2c) was validated using sera obtained weekly during 336 days from steers primed and boostered with BboR1A and/or BbiS1A on days 0 and 154, then compared with the immunofluorescent-antibody test (IFAT). Western blot (WB) protein analysis was used to confirm the specificity of the immune response to rMSA-2c. The sensitivity and specificity for ELISA(rMSA-2c) were 92 and 96% after the Babesia spp. priming and 88 and 73% after the boostering immunization, respectively. The sensitivity and specificity for IFAT were 99 and 90% after priming and 92 and 98% after boostering, respectively. Unlike IFAT, ELISA(rMSA-2c) detected a remarkable delayed booster response and a significant drop in specificity between 35 and 84 days after the booster immunization. Simultaneously, 87.5% of cattle boostered with B. bigemina showed cross-reactions in the ELISA(rMSA-2c), particularly between 63 and 77 days after the inoculation. A reaction against E. coli was observed, since bands of approximately 40 and/or 42kDa were detected using sera from cattle before and after Babesia spp. inoculations. ELISA(rMSA-2c) showed to be useful between 42 and 98 days after priming with Babesia spp. live vaccine to evaluate the success of infecting cattle. However, after boostering the test showed low specificity.

The search for vaccine candidates against bovine babesiosis caused by Babesia bovis is greatly focused on the identification of merozoite surface-exposed antigens that are widely conserved, functionally relevant and immunodominant in cattle protected against B. bovis infections. We have recently identified msa-2c, a member of the B. bovis variable merozoite surface antigen (VMSA) gene family, which in contrast to other members, appears to be highly conserved among geographically distant B. bovis strains. In this study, we further investigated the potential of the msa-2c gene product as diagnostic and vaccine candidate for bovine babesiosis. RT-PCR studies demonstrated that MSA-2c is transcribed in merozoites of the Argentine R1A strain. In addition, antibodies against R1A recombinant MSA-2c reacted in immunoblots with a single protein of approximately 30kDa in B. bovis merozoite extracts from both R1A and Australian "S" strains, demonstrating translation of this protein in these two strains and conservation of B-cell epitopes between them. These antibodies reacted with the cell surface of R1A merozoites in fixed immunofluorescence assays, indicating the surface localization of MSA-2c. This localization was confirmed by live immunofluorescence studies in two different strains, R1A and S2P. These results also demonstrate the conservation of MSA-2c surface-exposed B-cell epitopes between these two strains. Sera from cattle either naturally or experimentally infected with Argentine strains of B. bovis specifically recognized rMSA-2c in immunoblots, reinforcing the idea that B-cell epitopes in rMSA-2c are widely conserved among field strains of B. bovis. Furthermore, our results show that these B-cell epitopes are highly immunogenic, suggesting that MSA-2c may be a useful diagnostic tool for the detection of bovine babesiosis by B. bovis. Experimental vaccination of five bovines with rMSA-2c resulted in elicitation of high specific anti-rMSA-2c IgG titers, with similar amounts of IgG(1) and IgG(2) produced. Importantly, bovine anti-rMSA-2c antibodies were able to neutralize in vitro bovine erythrocyte invasion by R1A merozoites suggesting a significant functional role for MSA-2c. Taken together these results postulate MSA-2c as a candidate for the development of novel tools for improved control of bovine babesiosis.

Babesia bigemina infection of mature bovine erythrocytes results in new proteins specifically exposed on the parasitized cell surface. Monoclonal antibody (MAb) 64/32 binds a protein, designated p94, on B. bigemina -infected erythrocytes but not on either uninfected or B. bovis -parasitized erythrocytes. However, p94 was not encoded by B. bigemina and was not a parasite-modified erythrocyte membrane protein. In contrast, we showed that p94 could be eluted from the infected erythrocyte surface and was identified as specifically bound immunoglobulin M (IgM) heavy chain for the following reasons: (i) MAb 64/32 bound a reduced molecule of 94 kDa in both infected erythrocyte lysates and normal bovine serum; (ii) MAb 64/32 bound a 94-kDa molecule in reduced preparations of purified IgM; (iii) an anti-bovine μ heavy-chain MAb, BIg73, reacted specifically with the surface of infected erythrocytes and bound the 94-kDa molecule in lysates of infected erythrocytes, normal bovine serum, and purified IgM; and (iv) immunoprecipitation of infected erythrocyte lysates with MAb 64/32 depleted the 94-kDa antigen bound by anti-μ MAb BIg73 and vice versa. Binding of IgM to the infected erythrocyte surface was detected in vivo early in acute parasitemia and occurred during both the trophozoite and merozoite stages of intraerythrocytic parasitism. The common feature of IgM binding to the parasitized erythrocyte surface among otherwise genetically and antigenically distinct B. bigemina strains is suggestive of an advantageous role in parasite survival in vivo.

Members of the babesial rhoptry-associated protein 1 (RAP-1) family express surface-exposed B-cell epitopes and are candidate antigens for vaccine development. The relationship between rap-1 genomic polymorphism and surface-exposed B-cell epitope expression was analyzed by comparison of biological clones of Mexico strain Babesia bigemina and Babesia bovis with strains isolated in Argentina. Despite genomic polymorphism between strains, including sequences located within the open reading frame, defined RAP-1 B-cell surface epitopes and RAP-1 molecular size were conserved in both B. bovis and B. bigemina.

Background Cysteine proteases have been shown to be highly relevant for Apicomplexan parasites. In the case of Babesia bovis, a tick-transmitted hemoparasite of cattle, inhibitors of these enzymes were shown to hamper intraerythrocytic replication of the parasite, underscoring their importance for survival. Results Four papain-like cysteine proteases were found to be encoded by the B. bovis genome using the MEROPS database. One of them, the ortholog of Plasmodium falciparum falcipain-2, here named bovipain-2, was further characterized. Bovipain-2 is encoded in B. bovis chromosome 4 by an ORF of 1.3 kb, has a predicted molecular weight of 42 kDa, and is hydrophilic with the exception of a transmembrane region. It has orthologs in several other apicomplexans, and its predicted amino acid sequence shows a high degree of conservation among several B. bovis isolates from North and South America. Synteny studies demonstrated that the bovipain-2 gene has expanded in the genomes of two related piroplasmids, Theileria parva and T. annulata, into families of 6 and 7 clustered genes respectively. The bovipain-2 g ene is transcribed in in vitro cultured intra-erythrocyte forms of a virulent and an attenuated B. bovis strain from Argentina, and has no introns, as shown by RT-PCR followed by sequencing. Antibodies against a recombinant form of bovipain-2 recognized two parasite protein bands of 34 and 26 kDa, which coincide with the predicted sizes of the pro-peptidase and mature peptidase, respectively. Immunofluorescence studies showed an intracellular localization of bovipain-2 in the middle-rear region of in vitro cultured merozoites, as well as diffused in the cytoplasm of infected erythrocytes. Anti-bovipain-2 antibodies also reacted with B. bigemina-infected erythrocytes giving a similar pattern, which suggests cross-reactivity among these species. Antibodies in sera of two out of six B. bovis-experimentally infected bovines tested, reacted specifically with recombinant bovipain-2 in immunoblots, thus demonstrating expression and immunogenicity during bovine-infecting stages. Conclusions Overall, we present the characterization of bovipain-2 and demonstrate its in vitro and in vivo expression in virulent and attenuated strains. Given the involvement of apicomplexan cysteine proteases in essential parasite functions, bovipain-2 constitutes a new vaccine candidate and potential drug target for bovine babesiosis.