Nilgai antelope display no signs of infection upon experimental challenge with a virulent Babesia bovis strain.

BACKGROUND: Bovine babesiosis is caused by infection with the protozoal parasite Babesia bovis, which is transmitted by Rhipicephalus (Boophilus) spp. It can cause mortality rates up to 90% in immunologically naive Bos taurus cattle. In south Texas, R. (B.) microplus is known to infest nilgai antelope (Boselaphus tragocamelus); however, their susceptibility to infection with B. bovis and their role in the transmission of the parasite remain unknown. In this study, we challenged nilgai antelope with B. bovis and evaluated their susceptibility to infection. METHODS: Nilgai were needle inoculated with ≈108 B. bovis-parasitized erythrocytes (merozoites) or a homogenate of B. bovis-infected larval ticks (sporozoite) delivered intravenously. Bos taurus beef calves were inoculated in parallel, as this strain of B. bovis is lethal to cattle. Temperature and hematocrit were monitored daily over the course of each study, and whole blood was collected for molecular [polymerase chain reaction (PCR)] and serological [indirect enzyme-linked immunosorbent assay (ELISA)] diagnostic evaluation. Histological sections of nilgai cerebral tissue were examined for evidence of infection. Recipient bovine calves were sub-inoculated with blood from nilgai challenged with either stage of the parasite, and they were monitored for clinical signs of infection and evaluated by a PCR diagnostic assay. Red blood cells (RBCs) from prechallenged nilgai and B. taurus beef cattle were cultured with an in vitro B. bovis merozoite culture to examine colonization of the RBCs by the parasite. RESULTS: Nilgai did not display clinical signs of infection upon inoculation with either the merozoite or sporozoite stage of B. bovis. All nilgai were PCR-negative for the parasite, and they did not develop antibodies to B. bovis. No evidence of infection was detected in histological sections of nilgai tissues, and in vitro culture analysis indicated that the nilgai RBCs were not colonized by B. bovis merozoites. Cattle subinoculated with blood from challenged nilgai did not display clinical signs of infection, and they were PCR-negative up to 45 days after transfer. CONCLUSIONS: Nilgai do not appear to be susceptible to infection with a strain of B. bovis that is lethal to cattle. Tick control on these alternative hosts remains a critical priority, especially given their potential to disseminate ticks over long distances.

A conserved motif in the immune-subdominant RAP-1 related antigen of Babesia bovis contains a B-cell epitope recognized by antibodies from protected cattle.

Introduction: Babesia bovis, a tick-borne apicomplexan parasite causing bovine babesiosis, remains a significant threat worldwide, and improved and practical vaccines are needed. Previous studies defined the members of the rhoptry associated protein-1 (RAP-1), and the neutralization-sensitive rhoptry associated protein-1 related antigen (RRA) superfamily in B. bovis, as strong candidates for the development of subunit vaccines. Both RAP-1 and RRA share conservation of a group of 4 cysteines and amino acids motifs at the amino terminal end (NT) of these proteins. Methods and results: Sequence comparisons among the RRA sequences of several B. bovis strains and other Babesia spp parasites indicate a high level of conservation of a 15-amino acid (15-mer) motif located at the NT of the protein. BlastP searches indicate that the 15-mer motif is also present in adenylate cyclase, dynein, and other ATP binding proteins. AlphaFold2 structure predictions suggest partial exposure of the 15-mer on the surface of RRA of three distinct Babesia species. Antibodies in protected cattle recognize a synthetic peptide representing the 15-mer motif sequence in iELISA, and rabbit antibodies against the 15-mer react with the surface of free merozoites in immunofluorescence. Discussion and conclusion: The presence of the 15-mer-like regions in dynein and ATP-binding proteins provides a rationale for investigating possible functional roles for RRA. The demonstrated presence of a surface exposed B-cell epitope in the 15-mer motif of the B. bovis RRA, which is recognized by sera from protected bovines, supports its inclusion in future subunit epitope-based vaccines against B. bovis.

A small proportion of Zebu genetic background in crossbred calves may not be enough to improve resistance against natural bovine Babesia spp. infections.

The main objective of cattle breeders in tropical and subtropical regions is to acquire animals with taurine-productive traits adapted to the broad weather range of these regions. However, one of the main challenges on using taurine genetics in these areas is the high susceptibility of these animals to tick-borne diseases. Consequently, the present study evaluated from 10 November 2021-19 April 2022, the over 13 assessments, the Babesia bovis and Babesia bigemina DNA loads and the IgG anti-B. bovis and anti-B. bigemina levels in Angus (n = 17, 100% Taurine) and Ultrablack (n = 14, ∼82% taurine and 18% Zebu) calves. Data were analyzed using a multivariate mixed model with repeated measures of the same animal including the fixed effects of evaluation, genetic group, sex, Babesia spp., and their interactions. The repeatability values were estimated from the (co)variances matrix and expressed for each species. The correlations between the DNA loads (CNlog) and IgG titers (S/P) values for the two species were also estimated using the same model. Regarding the specific IgG antibody titers for both Babesia spp., no significant differences were observed between the two genetic groups. However, for B. bovis and B. bigemina DNA loads, Ultrablack calves presented significantly higher values than Angus calves. Under the conditions evaluated in this study, our findings suggest that the low percentage of Zebu genetic in the Ultrablack breed was insufficient to improve resistance against babesiosis. Further studies must demonstrate if the low percentages of Zebu genetics in Taurine breeds can modify the susceptibility to babesiosis infections.

Assessment of Babesia bovis 6cys A and 6cys B as components of transmission blocking vaccines for babesiosis.

BACKGROUND: Babesia bovis reproduces sexually in the gut of its tick vector Rhipicephalus microplus, which involves expression of 6cys A and 6cys B proteins. Members of the widely conserved 6cys superfamily are candidates for transmission blocking vaccines (TBV), but intricacies in the immunogenicity of the 6cys proteins in the related Plasmodium parasites required the identification of transmission blocking domains in these molecules for vaccine design. Hereby, the immunogenic efficacy of recombinant (r) B. bovis 6cys A and B proteins as a TBV formulation was studied. METHODS: The immunogenicity of r6cys A and 6cys B proteins expressed in a eukaryotic system was evaluated in a cattle immunization trial (3 immunized and 3 control calves). A B. bovis sexual stage induction in vitro inhibition assay to assess the ability of antibodies to block the production of sexual forms by the parasite was developed. RESULTS: Immunized cattle generated antibodies against r6cys A and r6cys B that were unable to block sexual reproduction of the parasite in ticks. Additionally, these antibodies also failed in recognizing native 6cys A and 6cys B and peptides representing 6cys A and 6cys B functional domains and in inhibiting the development of sexual forms in an in vitro induction system. In contrast, rabbit antibodies generated against synthetic peptides representing predicted B-cell epitopes of 6cys A and 6cys B recognized recombinant and native forms of both 6cys proteins as well as peptides representing 6cys A and 6cys B functional domains and were able to neutralize development of sexual forms of the parasite in vitro. CONCLUSIONS: These data, combined with similar work performed on Plasmodium 6cys proteins, indicate that an effective 6cys protein-based TBV against B. bovis will require identifying and targeting selected regions of proteins containing epitopes able to reduce transmission.

Genetic diversity of Babesia bovis studied longitudinally under natural transmission conditions in calves in the state of Rio de Janeiro, Brazil.

Serum and DNA samples from 15 naturally infected calves in Seropédica, Brazil, were obtained quarterly from birth to 12 months of age, in order to longitudinally evaluate their humoral immune response against Babesia bovis and the merozoite surface antigen diversity of B. bovis. Anti-B. bovis IgG antibodies were detected by an indirect fluorescent antibody test (IFAT) and enzyme-linked immunosorbent assay (ELISA). Using DNA amplification, sequencing and phylogenetic analysis, the genetic diversity of B. bovis was assessed based on the genes that encode merozoite surface antigens (MSA-1, MSA-2b and MSA-2c). The serological results demonstrated that up to six months of age, all the calves developed active immunity against B. bovis. Among the 75 DNA samples evaluated, 0, 3 and 5 sequences of the msa-1, msa-2b and msa-2c genes were obtained, respectively. The present study demonstrated that the msa-2b and msa-2c gene sequences amplified from blood DNA of B. bovis-positive calves were genetically diversified. These data emphasize the importance of conducting deeper studies on the genetic diversity of B. bovis in Brazil, in order to design diagnostic antigens and vaccines in the future.

A polymorphic CD4 epitope related to increased susceptibility to Babesia bovis in Canchim calves.

Different allelic forms of bovine CD4 were previously described in cattle and were also observed in Canchim calves examined in the present experiment. However, the functional relevance of these different CD4 phenotypes has not yet been investigated. CD4 + T helper cells are known to play a central role in immune control against Babesia bovis infection. Thus, our study aimed to compare the profiles of immune cells, specific antibody titers and blood infection levels measured by qPCR (quantitative polymerase chain reaction) in calves naturally infected with B. bovis, phenotyped as CD4- (absence of anti-CD4 staining), CD4 + (intermediate staining) or CD4 ++ (high staining). The CD4 mRNA precursor was also measured in these animals. Calves with the CD4- phenotype showed higher amounts of B. bovis DNA in blood samples, compared to the other CD4 phenotypes. It was also observed that these calves with higher levels of infection had lower amounts of natural killer cells and higher expression of the CD4 gene, which can be interpreted as a compensation for the failure of the altered CD4 receptor to recognize relevant B. bovis epitopes.

Babesia bovis Rad51 ortholog influences switching of ves genes but is not essential for segmental gene conversion in antigenic variation.

The tick-borne apicomplexan parasite, Babesia bovis, a highly persistent bovine pathogen, expresses VESA1 proteins on the infected erythrocyte surface to mediate cytoadhesion. The cytoadhesion ligand, VESA1, which protects the parasite from splenic passage, is itself protected from a host immune response by rapid antigenic variation. B. bovis relies upon segmental gene conversion (SGC) as a major mechanism to vary VESA1 structure. Gene conversion has been considered a form of homologous recombination (HR), a process for which Rad51 proteins are considered pivotal components. This could make BbRad51 a choice target for development of inhibitors that both interfere with parasite genome integrity and disrupt HR-dependent antigenic variation. Previously, we knocked out the Bbrad51 gene from the B. bovis haploid genome, resulting in a phenotype of sensitivity to methylmethane sulfonate (MMS) and apparent loss of HR-dependent integration of exogenous DNA. In a further characterization of BbRad51, we demonstrate here that ΔBbrad51 parasites are not more sensitive than wild-type to DNA damage induced by γ-irradiation, and repair their genome with similar kinetics. To assess the need for BbRad51 in SGC, RT-PCR was used to observe alterations to a highly variant region of ves1α transcripts over time. Mapping of these amplicons to the genome revealed a significant reduction of in situ transcriptional switching (isTS) among ves loci, but not cessation. By combining existing pipelines for analysis of the amplicons, we demonstrate that SGC continues unabated in ΔBbrad51 parasites, albeit at an overall reduced rate, and a reduction in SGC tract lengths was observed. By contrast, no differences were observed in the lengths of homologous sequences at which recombination occurred. These results indicate that, whereas BbRad51 is not essential to babesial antigenic variation, it influences epigenetic control of ves loci, and its absence significantly reduces successful variation. These results necessitate a reconsideration of the likely enzymatic mechanism(s) underlying SGC and suggest the existence of additional targets for development of small molecule inhibitors.

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

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

Identification of conserved peptides containing B-cell epitopes of Babesia bovis AMA-1 and their potential as diagnostics candidates.

The apical membrane antigen 1 (AMA-1) is a protein of the micronemes that is present in all organisms of the phylum Apicomplexa; it has been shown that AMA-1 plays an essential role for parasite invasion to target cells. It has been reported that AMA-1 is conserved among different isolates of Babesia; however, it is unknown whether the protein contains conserved B-cell epitopes and whether these epitopes are recognized by antibodies from cattle in endemic areas. In this research, using an in silico analysis, four peptides were designed containing exposed and conserved linear B-cell epitopes from the extracellular region of Babesia bovis AMA-1. The selected peptides were chemically synthesized, and then each peptide was emulsified and used to immunize two bovines per peptide. The antibodies produced against these peptides were able to recognize intra-erythrocytic parasites in an IFAT, except peptide 4, which was insoluble. The synthetic peptides were covalently fixed to the wells of an ELISA plate and incubated with sera from B. bovis naturally infected cattle. Peptides P2AMA and P3AMA were recognized by the sera of naturally infected cattle from different regions of Mexico. Statistical analysis showed that the ELISA test for peptides P2AMA and P3AMA had a concordance of 91.2% and 61.1% compared to the IFAT, a sensitivity of 94.56% and 71.74%, and a specificity of 76.19% and 14.2%, respectively. The presence of antibodies in bovine sera from endemic areas that bind to the identified peptides indicates that AMA-1 from B. bovis has conserved B-cell epitopes involved in the immune response under natural conditions. However, to propose their use as vaccine or diagnostics candidates, a further characterization of the humoral immune response elicited in cattle by these peptides is needed.

Highly sensitive nested PCR and rapid immunochromatographic detection of Babesia bovis and Babesia bigemina infection in a cattle herd with acute clinical and fatal cases in Argentina.

Bovine babesiosis is a tick-transmitted haemoparasitic disease caused by Babesia bovis and B. bigemina affecting cattle of tropical and subtropical regions around the world. Pathogens are transmitted by the tick vector Rhipicephalus microplus displaying a widespread distribution in northeastern Argentina. The disease is characterized by significant animal morbidity and mortality resulting in considerable economic loss. In this study, B. bovis and B. bigemina infection was investigated in a cattle herd of 150 adult bovines of pure Braford breed raised in a tick-hyperendemic field using molecular and serum antibody tests. A highly sensitive nested polymerase chain reaction (nPCR) assay targeting a species-specific region of the apocytochrome b gene resulted in direct B. bovis and B. bigemina detection in 27.3% and 54.7% of bovines, respectively. A recently developed immunochromatographic strip test (ICT) based on recombinant forms of spherical body protein 4 and the C-terminal region of rhoptry-associated protein 1 showed that 71.3% and 89.3% of bovines were seropositive for B. bovis and B. bigemina, respectively. The mixed infection rate as observed by direct (19.3%) and indirect detection (65.3%) coincided with those expected, respectively. Importantly, four months after sampling, nine bovines of the studied herd showed clinical signs of bovine babesiosis of which six animals eventually died. Microscopic detection of infected erythrocytes in Giemsa-stained blood smears confirmed B. bovis infection. Our study demonstrates that although animals showed a relatively high and very high rate of immunity against infection with B. bovis (71.3%) and B. bigemina (89.3%) parasites, respectively, clinical cases and fatalities due to the infection with B. bovis were observed. It is proposed that the most adequate control measure in the studied epidemiological situation is to vaccinate animals to prevent losses and/or an outbreak of bovine babesiosis.

Genetic diversity of Babesia bovis studied longitudinally under natural transmission conditions in calves in the state of Rio de Janeiro, Brazil / Diversidade genética de Babesia bovis estudada longitudinalmente em condições de transmissão natural entre bezerros no estado do Rio de Janeiro, Brasil

Abstract Serum and DNA samples from 15 naturally infected calves in Seropédica, Brazil, were obtained quarterly from birth to 12 months of age, in order to longitudinally evaluate their humoral immune response against Babesia bovis and the merozoite surface antigen diversity of B. bovis. Anti-B. bovis IgG antibodies were detected by an indirect fluorescent antibody test (IFAT) and enzyme-linked immunosorbent assay (ELISA). Using DNA amplification, sequencing and phylogenetic analysis, the genetic diversity of B. bovis was assessed based on the genes that encode merozoite surface antigens (MSA-1, MSA-2b and MSA-2c). The serological results demonstrated that up to six months of age, all the calves developed active immunity against B. bovis. Among the 75 DNA samples evaluated, 0, 3 and 5 sequences of the msa-1, msa-2b and msa-2c genes were obtained, respectively. The present study demonstrated that the msa-2b and msa-2c gene sequences amplified from blood DNA of B. bovis-positive calves were genetically diversified. These data emphasize the importance of conducting deeper studies on the genetic diversity of B. bovis in Brazil, in order to design diagnostic antigens and vaccines in the future.

Resumo Para avaliar longitudinalmente a resposta imune humoral anti-B. bovis e a diversidade genética de antígenos de superfície de merozoítos de B. bovis, entre bezerros naturalmente infectados em Seropédica, Brasil, amostras de soro e DNA de 15 bezerros foram obtidas trimestralmente, desde o nascimento até 12 meses de idade. Anticorpos IgG para B. bovis foram detectados pelos testes de Imunofluorescência Indireta e Ensaio de Imunoadsorção Enzimático Indireto. Usando-se amplificação de DNA, sequenciamento e análises filogenéticas, a diversidade genética de B. bovis, com base nos genes que codificam antígenos de superfície de merozoítos (MSA-1, MSA-2b e MSA-2c) foi investigada. Os resultados da sorologia demonstraram que, até os seis meses de idade, todos os bezerros desenvolveram imunidade ativa contra B. bovis. Entre as 75 amostras de DNA avaliadas, foram obtidas 0, 3 e 5 sequências dos genes msa-1, msa-2b e msa-2c. O presente estudo demonstrou que sequências dos genes msa-2b e msa-2c amplificadas a partir de amostras de sangue positivas para B. bovis de bezerros de Seropédica, foram geneticamente distintas. O presente trabalho realça a importância de se realizar estudos aprofundados sobre a diversidade genética de B. bovis no Brasil, objetivando o desenvolvimento de antígenos para o diagnóstico e vacinas no futuro.

Cocktail Babesia bovis antigens for global detection of Babesia bovis infection in cattle.

The diagnostic performance of a cocktail formula consisting of two Babesia (B.) bovis recombinant proteins, including spherical body protein 1 (BbSBP-1) and spherical body protein 4 (BbSBP-4), was evaluated in the present study for the global detection of B. bovis infection in cattle and for the differentiation between B. bovis and B. bigemina infections. The efficacy and the practicality of the rBbSBP-1 and rBbSBP-4 cocktail formula for differentiation between the infection caused by both parasites were assessed using indirect enzyme-linked immunosorbent assay (iELISA) with serum samples collected from cattle experimentally infected by B. bovis (n = 33) or B. bigemina (n = 30). Cocktail antigen exhibited the highest optical density (OD) values with B. bovis-infected sera and the lowest OD values with normal bovine sera or B. bigemina-infected sera in comparison with the single antigen. A total of 581 field serum samples collected from four countries with known B. bovis endemicity: Ghana (n = 154), Egypt (n = 162), Thailand (n = 96), and South Africa (n = 169) were screened also in the current study using iELISA and the results were compared to those of indirect fluorescent antibody test (IFAT) as a reference. A cocktail formula (rBbSBP-1 and rBbSBP-4) exhibited the highest concordance rate (89.90%) and kappa value (0.73). The obtained results revealed the reliability of the rBbSBP-1 and rBbSBP-4 cocktail antigen for the detection of specific antibodies to B. bovis in cattle and demonstrated the usefulness of cocktail antigen for differentiation between B. bovis and B. bigemina infections compared with the single antigen in cattle, which will be useful for epidemiological surveys and control of bovine babesiosis.

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.

Babesia bovis RON2 contains conserved B-cell epitopes that induce an invasion-blocking humoral immune response in immunized cattle.

BACKGROUND: Babesia bovis belongs to the phylum Apicomplexa and is the major causal agent of bovine babesiosis, the most important veterinary disease transmitted by arthropods. In apicomplexan parasites, the interaction between AMA1 and RON2 is necessary for the invasion process, and it is a target for vaccine development. In B. bovis, the existence of AMA1 has already been reported; however, the presence of a homolog of RON2 is unknown. The aim of this study was to characterize RON2 in B. bovis. RESULTS: The B. bovis ron2 gene has a similar synteny with the orthologous gene in the B. bigemina genome. The entire ron2 gene was sequenced from different B. bovis strains showing > 99% similarity at the amino acid and nucleotide level among all the sequences obtained, including the characteristic CLAG domain for cytoadherence in the amino acid sequence, as is described in other Apicomplexa. The in silico transcription analysis showed similar levels of transcription between attenuated and virulent B. bovis strains, and expression of RON2 was confirmed by western blot in the B. bovis T3Bo virulent strain. Four conserved peptides, containing predicted B-cell epitopes in hydrophilic regions of the protein, were designed and chemically synthesized. The humoral immune response generated by the synthetic peptides was characterized in bovines, showing that anti-RON2 antibodies against peptides recognized intraerythrocytic merozoites of B. bovis. Only peptides P2 and P3 generated partially neutralizing antibodies that had an inhibitory effect of 28.10% and 21.42%, respectively, on the invasion process of B. bovis in bovine erythrocytes. Consistently, this effect is additive since inhibition increased to 42.09% when the antibodies were evaluated together. Finally, P2 and P3 peptides were also recognized by 83.33% and 87.77%, respectively, of naturally infected cattle from endemic areas. CONCLUSIONS: The data support RON2 as a novel B. bovis vaccine candidate antigen that contains conserved B-cell epitopes that elicit partially neutralizing antibodies.

Mitigated clinical disease in water buffaloes experimentally infected with Babesia bovis.

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 108 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.

Bovine babesiosis: Cattle protected in the field with a frozen vaccine containing Babesia bovis and Babesia bigemina cultured in vitro with a serum-free medium.

An attenuated live vaccine containing Babesia bovis and B. bigemina cultured in vitro with a serum-free medium was assessed for its clinical protection conferred of naïve cattle, under natural tick-challenge in a high endemicity zone to Babesia spp. Three groups of six animals were treated as follows: group I (GI) received a vaccine derived from parasites cultured with a free-serum medium; group II (GII) were immunized with the standard vaccine, with parasites cultured in a medium supplemented with 40% (v/v) bovine serum; and a control group (GIII) inoculated with non-infected bovine erythrocytes. Inocula were administered by IM route. Experimental animals were kept during 23days after vaccination in a cattle farm free of ticks and Babesia spp. Thereafter, cattle were moved to a high endemicity farm for natural exposure to Babesia spp. transmitted by Rhipicephalus microplus ticks. Protection against clinical babesiosis was observed in bovines belonging to GI (100%) and GII (83.33%), while the control animals (GIII) were not protected, and showed severe clinical signs, closely related to babesiosis, were observed for at least three consecutive days during the challenge. These were fever, anemia, which were measured simultaneously, and circulating parasites were detected by optic light microscopy. All cattle showed B. bovis and B. bigemina in stained blood films during the challenge; B. bovis antibody titers were higher than those to B. bigemina in GI and GII, and lower titers were determined in GIII. The protective capacity of the vaccine derived from B. bovis and B. bigemina cultured in vitro in a serum-free medium was demonstrated.

Longitudinal evaluation of humoral immune response and merozoite surface antigen diversity in calves naturally infected with Babesia bovis, in São Paulo, Brazil.

Babesiosis is an economically important infectious disease affecting cattle worldwide. In order to longitudinally evaluate the humoral immune response against Babesia bovis and the merozoite surface antigen diversity of B. bovis among naturally infected calves in Taiaçu, Brazil, serum and DNA samples from 15 calves were obtained quarterly, from their birth to 12 months of age. Anti-B. bovis IgG antibodies were detected by means of the indirect fluorescent antibody test (IFAT) and enzyme-linked immunosorbent assay (ELISA). The polymerase chain reaction (PCR) was used to investigate the genetic diversity of B. bovis, based on the genes that encode merozoite surface antigens (MSA-1, MSA-2b and MSA-2c). The serological results demonstrated that up to six months of age, all the calves developed active immunity against B. bovis. Among the 75 DNA samples evaluated, 2, 4 and 5 sequences of the genes msa-1, msa-2b and msa-2c were obtained. The present study demonstrated that the msa-1 and msa-2b genes sequences amplified from blood DNA of calves positive to B. bovis from Taiaçu were genetically distinct, and that msa-2c was conserved. All animals were serologically positive to ELISA and IFAT, which used full repertoire of parasite antigens in despite of the genetic diversity of MSAs.

Longitudinal evaluation of humoral immune response and merozoite surface antigen diversity in calves naturally infected with Babesia bovis, in São Paulo, Brazil / Avaliação longitudinal da resposta immune humoral e diversidade de antígenos de superfície de merozoítos em bezerros naturalmente infectados com Babesia bovis, em São Paulo, Brasil

Abstract Babesiosis is an economically important infectious disease affecting cattle worldwide. In order to longitudinally evaluate the humoral immune response against Babesia bovis and the merozoite surface antigen diversity of B. bovis among naturally infected calves in Taiaçu, Brazil, serum and DNA samples from 15 calves were obtained quarterly, from their birth to 12 months of age. Anti-B. bovis IgG antibodies were detected by means of the indirect fluorescent antibody test (IFAT) and enzyme-linked immunosorbent assay (ELISA). The polymerase chain reaction (PCR) was used to investigate the genetic diversity of B. bovis, based on the genes that encode merozoite surface antigens (MSA-1, MSA-2b and MSA-2c). The serological results demonstrated that up to six months of age, all the calves developed active immunity against B. bovis. Among the 75 DNA samples evaluated, 2, 4 and 5 sequences of the genes msa-1, msa-2b and msa-2c were obtained. The present study demonstrated that the msa-1 and msa-2b genes sequences amplified from blood DNA of calves positive to B. bovis from Taiaçu were genetically distinct, and that msa-2c was conserved. All animals were serologically positive to ELISA and IFAT, which used full repertoire of parasite antigens in despite of the genetic diversity of MSAs.

Resumo A babesiose é uma doença infecciosa economicamente importante que afeta o gado bovino em todo o mundo. Para avaliar longitudinalmente a resposta imune humoral contra B. bovis e a diversidade genética de antígenos de superfície de merozoítos de B. bovis, entre bezerros naturalmente infectados em Taiaçu, Brasil, amostras de soro e DNA de 15 bezerros, foram obtidos trimestralmente, desde o nascimento até aos 12 meses de idade. Os anticorpos IgG para B. bovis foram detectados pelos testes de Imunofluorescência Indireta e Ensaio de Imunoadsorção Enzimático Indireto. A Reação em Cadeia da Polimerase foi utilizada para investigar a diversidade genética de B. bovis, com base em genes que codificam antígenos de superfície de merozoítos (MSA-1, MSA-2b e MSA-2c). Os resultados da sorologia demonstraram que até seis meses de idade todos os bezerros desenvolveram imunidade ativa contra B. bovis. Entre as 75 amostras de DNA avaliadas, foram obtidas 2, 4 e 5 sequências dos genes msa-1, msa-2b e msa-2c. O presente trabalho demonstrou que as sequências dos genes msa-1 e msa-2b amplificadas do DNA do sangue de amostras positivas a B. bovis de bezerros de Taiaçu foram geneticamente distintas, e msa-2c conservadas. Todos os animais foram soropositivos ao ELISA e ao IFAT, os quais utilizaram o repertório completo de antígenos parasitários, apesar da diversidade genética dos MSAs.

Molecular and serological detection of bovine babesiosis in Indonesia.

BACKGROUND: Bovine babesiosis, mainly caused by Babesia bovis and B. bigemina, is a huge threat to the livestock industry. In Indonesia, the current distribution of the disease is unknown due to a lack of scientific study. METHODS: In the present study, 487 blood samples were collected from cattle with different breeding and age groups in a broad geographical area across the archipelago. The presence of antibodies and current infections of B. bovis and B. bigemina were determined using enzyme-linked immunosorbent assay (ELISA), immunochromatographic test (ICT), and nested PCR (nPCR) targeting B. bovis SBP-4 and B. bigemina RAP-1a genes. Sequence analysis was performed to the amplicon of B. bovis SBP-4, B. bigemina RAP-1a, and internal transcribed spacer (ITS) region of ribosomal RNA of both Babesia species. RESULTS: In total, B. bovis positives were detected by ELISA, single-ICT, dual-ICT and nPCR in 340 (69.8%), 317 (65.1%), 307 (63.0%) and 247 (50.7%) samples, respectively. For B. bigemina, the positive samples were detected in 134 (27.5%), 130 (26.7%), 127 (26.1%) and 93 (19.1%), respectively. Furthermore, mixed infections were found in 125 (25.7%), 113 (23.2%), 109 (22.4%) and 52 (10.7%) samples, respectively, which occurred only by chance and were not influenced by additional factors. The obtained nucleotide sequences of B. bovis SBP-4 and B. bigemina RAP-1a genes showed a high homology with other isolates from different countries. Further nucleotide sequence analysis using ITS region showed a great genetic diversity of B. bovis isolates among sampling locations; a lower diversity was found in B. bigemina ITS isolates. CONCLUSIONS: These data revealed the current distribution of B. bovis and B. bigemina infection in cattle in Indonesia. The rate of infection varied among sampling locations, cattle breeds and age groups. Furthermore, B. bovis ITS isolates from Indonesia were found to be more genetically diverse than B. bigemina ITS isolates. The data presented in this study are necessary to develop an effective strategy for controlling the disease in the country.

Babesia bovis BOV57, a Theileria parva P67 homolog, is an invasion-related, neutralization-sensitive antigen.

Babesia bovis BOV57, which is a homolog of the Theileria parva vaccine candidate antigen P67, is expressed in both the tick and blood stages of the life cycle of this parasite. However, the vaccine potential of BOV57 remained to be investigated. In the present study, we generated recombinant BOV57 (rBOV57) and prepared polyclonal antibodies against rBOV57 in mice and rabbits. Indirect immunofluorescence assays conducted with the mouse anti-rBOV57 antibody demonstrated that BOV57 localized at the apical end of paired merozoites in infected bovine red blood cells, whereas the antigen was found in the parasite membrane around the apical end of intraerythrocytic single and extracellular merozoites. In an invasion-inhibition assay, the rabbit anti-rBOV57 antibody potentially inhibited RBC invasion of B. bovis merozoites in vitro. In addition, the invasion inhibition mediated by rabbit anti-rBOV57 antibody resulted in a reduced growth rate of B. bovis in the in vitro culture. These findings indicated that B. bovis BOV57 plays a critical role in the invasion of merozoites into red blood cells, suggesting its potential as a subunit vaccine candidate against B. bovis infection in cattle. Furthermore, we analyzed the genetic diversity of bov57 gene sequences isolated from Sri Lanka, Mongolia, the Philippines, and Vietnam. The bov57 gene sequences derived from Mongolia, the Philippines, and Vietnam were conserved, whereas insertion and/or deletion mutations resulted in sequence diversity among the Sri Lankan samples. In summary, BOV57 is an invasion-related, neutralization-sensitive antigen encoded by the bov57 gene, which displays higher sequence diversity than previously reported.