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.

Babesiosis as a potential threat for bovine production in China.

Babesiosis is a tick-borne disease with global impact caused by parasites of the phylum Apicomplexa, genus Babesia. Typically, acute bovine babesiosis (BB) is characterized by fever, anemia, hemoglobinuria, and high mortality. Surviving animals remain persistently infected and become reservoirs for parasite transmission. Bovids in China can be infected by one or more Babesia species endemic to the country, including B. bovis, B. bigemina, B. orientalis, B. ovata, B. major, B. motasi, B. U sp. Kashi and B. venatorum. The latter may pose a zoonotic risk. Occurrence of this wide diversity of Babesia species in China may be due to a combination of favorable ecological factors, such as the presence of multiple tick vectors, including Rhipicephalus and Hyalomma, the coexistence of susceptible bovid species, such as domestic cattle, yaks, and water buffalo, and the lack of efficient measures of tick control. BB is currently widespread in several regions of the country and a limiting factor for cattle production. While some areas appear to have enzootic stability, others have considerable cattle mortality. Research is needed to devise solutions to the challenges posed by uncontrolled BB. Critical research gaps include risk assessment for cattle residing in endemic areas, understanding factors involved in endemic stability, evaluation of parasite diversity and pathogenicity of regional Babesia species, and estimation of whether and how BB should be controlled in China. Research should allow the design of comprehensive interventions to improve cattle production, diminish the risk of human infections, and increase the availability of affordable animal protein for human consumption in China and worldwide. In this review, we describe the current state of BB with reference to the diversity of hosts, vectors, and parasite species in China. We also discuss the unique risks and knowledge gaps that should be taken into consideration for future Babesia research and control strategies.

Genomic Study of Babesia bovis Infection Level and Its Association With Tick Count in Hereford and Braford Cattle.

Bovine babesiosis is a tick-borne disease caused by intraerythrocytic protozoa and leads to substantial economic losses for the livestock industry throughout the world. Babesia bovis is considered the most pathogenic species, which causes bovine babesiosis in Brazil. Genomic data could be used to evaluate the viability of improving resistance against B. bovis infection level (IB) through genomic selection, and, for that, knowledge of genetic parameters is needed. Furthermore, genome-wide association studies (GWAS) could be conducted to provide a better understanding of the genetic basis of the host response to B. bovis infection. No previous work in quantitative genetics of B. bovis infection was found. Thus, the objective of this study was to estimate the genetic correlation between IB and tick count (TC), evaluate predictive ability and applicability of genomic selection, and perform GWAS in Hereford and Braford cattle. The single-step genomic best linear unbiased prediction method was used, which allows the estimation of both breeding values and marker effects. Standard phenotyping was conducted for both traits. IB quantifications from the blood of 1,858 animals were carried using quantitative PCR assays. For TC, one to three subsequent tick counts were performed by manually counting adult female ticks on one side of each animal's body that was naturally exposed to ticks. Animals were genotyped using the Illumina BovineSNP50 panel. The posterior mean of IB heritability, estimated by the Bayesian animal model in a bivariate analysis, was low (0.10), and the estimations of genetic correlation between IB and TC were also low (0.15). The cross-validation genomic prediction accuracy for IB ranged from 0.18 to 0.35 and from 0.29 to 0.32 using k-means and random clustering, respectively, suggesting that genomic predictions could be used as a tool to improve genetics for IB, especially if a larger training population is developed. The top 10 single nucleotide polymorphisms from the GWAS explained 5.04% of total genetic variance for IB, which were located on chromosomes 1, 2, 5, 6, 12, 17, 18, 16, 24, and 26. Some candidate genes participate in immunity system pathways indicating that those genes are involved in resistance to B. bovis in cattle. Although the genetic correlation between IB and TC was weak, some candidate genes for IB were also reported in tick infestation studies, and they were also involved in biological resistance processes. This study contributes to improving genetic knowledge regarding infection by B. bovis in cattle.

Novel Babesia bovis exported proteins that modify properties of infected red blood cells.

Babesia bovis causes a pathogenic form of babesiosis in cattle. Following invasion of red blood cells (RBCs) the parasite extensively modifies host cell structural and mechanical properties via the export of numerous proteins. Despite their crucial role in virulence and pathogenesis, such proteins have not been comprehensively characterized in B. bovis. Here we describe the surface biotinylation of infected RBCs (iRBCs), followed by proteomic analysis. We describe a multigene family (mtm) that encodes predicted multi-transmembrane integral membrane proteins which are exported and expressed on the surface of iRBCs. One mtm gene was downregulated in blasticidin-S (BS) resistant parasites, suggesting an association with BS uptake. Induced knockdown of a novel exported protein encoded by BBOV_III004280, named VESA export-associated protein (BbVEAP), resulted in a decreased growth rate, reduced RBC surface ridge numbers, mis-localized VESA1, and abrogated cytoadhesion to endothelial cells, suggesting that BbVEAP is a novel virulence factor for B. bovis.

Use of molecular markers can help to understand the genetic diversity of Babesia bovis.

Cattle babesiosis is a tick-borne disease responsible for significant losses for the livestock industries in tropical areas of the world. These piroplasms are under constant control of the host immune system, which lead to a strong selective pressure for arising more virulent or attenuated phenotypes. Aiming to better understand the most critical genetic modifications in Babesia bovis genome, related to virulence, an in silico analysis was performed using DNA sequences from GenBank. Fourteen genes (sbp-2, sbp-4, trap, msa-1, msa-2b, msa-2c, Bv80 (or Bb-1), 18S rRNA, acs-1, ama-1, ß-tub, cp-2, p0, rap-1a) related to parasite infection and immunogenicity and ITS region were selected for alignment and comparison of several isolates of Babesia bovis from different geographic regions around the world. Among the 15 genes selected for the study of diversity, only 7 genes (sbp-2, sbp-4, trap, msa-1, msa-2b, msa-2c, Bv80) and the ITS region presented sufficient genetic variation for the studies of phylogeny. Despite this genetic diversity observed into groups, there was not sufficient information available to associate molecular markers with virulence of isolates. However, some genetic groups no were correlated with geographic region what could indicate some typical evolutionary characteristics in the relation between parasite-host. Further studies using these genes in herds presenting diverse clinical conditions are required. The better understanding of evolutionary mechanisms of the parasite may contribute to improve prophylactic and therapeutic measures. In this way, we suggest that genes used in our study are potential markers of virulence and attenuation and have to be analyzed with the use of sequences from animals that present clinical signs of babesiosis and asymptomatic carriers.

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.

Prevalence, risk factors, and genetic diversity of veterinary important tick-borne pathogens in cattle from Rhipicephalus microplus-invaded and non-invaded areas of Benin.

Babesiosis, theileriosis, anaplasmosis, and heartwater are tick-borne diseases (TBD) that threaten livestock production in sub-Saharan Africa including Benin. This country has been faced with an invasion of Rhipicephalus microplus, a major vector for babesiosis, theileriosis, and anaplasmosis over the last decade. Yet, data on TBD and the impact of the invasive ticks are lacking, making risk level evaluation and disease control arduous. In this study, epidemiological features of Babesia bovis, B. bigemina, Theileria spp., Anaplasma marginale and Ehrlichia ruminantium infections in Benin cattle were investigated in R. microplus-invaded and non-invaded areas. Detection of pathogens was based on species-specific PCR assays and resulting data were used to identify risk factors. Genetic diversity and phylogenies were then evaluated using several markers. Out of 207 samples examined, 170 (82.1%), 109 (52.7%), 42 (20.3%) 24 (11.6%) and 1 (0.5%) were positive for T. mutans, A. marginale, B. bigemina, B. bovis and E. ruminantium, respectively. Animal gender (for B. bovis), exposure to R. microplus (for B. bigemina and A. marginale), animal age (for B. bigemina and A. marginale) and cattle breed and/or antiprotozoal treatment (for T. mutants) significantly modulated pathogen occurrence. In addition, R. microplus exposure was significantly related to co-infection patterns and cases of clinical theileriosis and/or anaplasmosis were recorded among cattle highly exposed to the tick. In the genetic characterization, Theileria spp. and E. ruminantium sequences were conserved. Babesia spp. and A. marginale, however, showed high sequence polymorphisms that indicate the presence of several strains and may be linked to R. microplus invasion. Taken together, these results ascertain the endemicity of tick-borne infections in Benin and suggest that the characteristics of Babesia spp. and A. marginale infections in R. microplus-invaded and non-invaded areas are different.

Involvement of TLR6 in the induction of COX-2, PGE2 and IL-10 in macrophages by lipids from virulent S2P and attenuated R1A Babesia bovis strains.

Toll like receptors (TLRs) are involved in the modulation of diverse host genes expression through a complex network of signalling events that allow for an appropriate response to a microbial pathogen. In the present work we used TLR6KO mice in order to study the role of TLR6 in the immune discrimination of lipids from two Babesia bovis strains, attenuated R1A (LA) and virulent S2P (LV), and the consequent macrophage activation. We demonstrated that TLR6 is required for lipid body induction in murine peritoneal macrophages by both LA and LV. Interestingly, as regards IL-10 and COX-2/PGE2 pathway induction by LA and LV, we observed differences in the biological effects produced by these lipid extracts. Our results indicate a role of TLR6 in the down-modulation of these immunoregulators only in the case of LA, whereas this receptor was not implicated in pro-inflammatory TNFα, IL-6 and KC release induced by LA. Remarkably, LV did not exert the down-modulatory effect observed for LA, supporting the notion that LA and LV possess different lipid composition that could correlate with the polar pathogenic effect of both B. bovis strains.

Co-transmission of the non-transmissible South African Babesia bovis S24 vaccine strain during mixed infection with a field isolate.

The South African Babesia bovis live blood vaccine, originating from a field isolate attenuated by 23 serial syringe passages in splenectomized calves, has lost the ability to infect the natural vector Rhipicephalus (Boophilus) microplus. In this study, infection with mixed parasites from the vaccine strain and a field isolate, resulted in transmission of both genotype populations. Comparing the field isolate and transmitted combination indicated no significant difference in their virulence, while challenge of vaccinated cattle with these isolates showed the ability of the vaccine to protect against both. Limiting dilution of the transmitted combination, followed by infection of splenectomized cattle (n=34) yielded no single infections for the vaccine strain genotype, seven clonal lines of the field isolate and one mixture of vaccine strain and field isolate. Only one of two field isolate clonal lines selected for vector transmission study was transmitted. Showing that B. bovis isolates can contain both tick transmissible and non-transmissible subpopulations. The findings of this study also indicate the probability of vaccine co-infection transmission occurring in the field, which may result in new genotype populations of B. bovis. However, the impact of this recombination with field isolates is considered negligible since a genotypically diverse population of B. bovis is already present in South Africa.

Tick passage results in enhanced attenuation of Babesia bovis.

Serial blood passage of virulent Babesia bovis in splenectomized cattle results in attenuated derivatives that do not cause neurologic disease. Tick transmissibility can be lost with attenuation, but when retained, attenuated B. bovis can revert to virulence following tick passage. This study provides data showing that tick passage of the partially attenuated B. bovis T2Bo derivative strain further decreased virulence compared with intravenous inoculation of the same strain in infected animals. Ticks that acquired virulent or attenuated parasites by feeding on infected cattle were transmission fed on naive, splenectomized animals. While there was no significant difference between groups in the number of parasites in the midgut, hemolymph, or eggs of replete female ticks after acquisition feeding, animals infected with the attenuated parasites after tick transmission showed no clinical signs of babesiosis, unlike those receiving intravenous challenge with the same attenuated strain prior to tick passage. Additionally, there were significantly fewer parasites in blood and tissues of animals infected with tick-passaged attenuated parasites. Sequencing analysis of select B. bovis genes before and after tick passage showed significant differences in parasite genotypes in both peripheral blood and cerebral samples. These results provide evidence that not only is tick transmissibility retained by the attenuated T2Bo strain, but also it results in enhanced attenuation and is accompanied by expansion of parasite subpopulations during tick passage that may be associated with the change in disease phenotype.

Comparative transcriptome analysis of geographically distinct virulent and attenuated Babesia bovis strains reveals similar gene expression changes through attenuation.

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.

Loss of neurovirulence is associated with reduction of cerebral capillary sequestration during acute Babesia bovis infection.

BACKGROUND: Severe neurological signs that develop during acute infection by virulent strains of Babesia bovis are associated with sequestration of infected erythrocytes in cerebral capillaries. Serial passage of virulent strains in cattle results in attenuated derivatives that do not cause neurologic disease. We evaluated whether serial passage also results in a loss of cerebral capillary sequestration by examining brain biopsies during acute disease and at necropsy. FINDINGS: Cerebral biopsies of spleen intact calves inoculated intravenously with a virulent or attenuated strain pair of B. bovis were evaluated for capillary sequestration at the onset of babesiosis and during severe disease. In calves infected with the virulent strain, there was a significant increase in sequestration between the first and second biopsy timepoint. The attenuated strain was still capable of sequestration, but at a reduced level, and did not change significantly between the first and second biopsy. Necropsy examination confirmed the second biopsy results and demonstrated that sequestration identified at necropsy reflects pathologic changes occurring in live animals. CONCLUSIONS: Loss of neurovirulence after serial in vivo passage of the highly virulent T2Bo strain of B. bovis in splenectomized animals is associated with a significant reduction of cerebral capillary sequestration. Previous genomic analysis of this and two other strain pairs suggests that this observation could be related to genomic complexity, particularly of the ves gene family, rather than consistent gene specific differences. Additional experiments will examine whether differential gene expression of ves genes is also associated with reduced cerebral sequestration and neurovirulence in attenuated strains.

Babesia bovis: lipids from virulent S2P and attenuated R1A strains trigger differential signalling and inflammatory responses in bovine macrophages.

The intra-erythrocytic protozoan Babesia bovis is an economically important pathogen that causes an acute and often fatal infection in adult cattle. Babesiosis limitation depends on the early activation of macrophages, essential cells of the host innate immunity, which can generate an inflammatory response mediated by cytokines and nitric oxide (NO). Herein, we demonstrate in bovine macrophages that lipids from B. bovis attenuated R1A strain (LA) produced a stronger NO release, an early TNFα mRNA induction and 2-fold higher IL-12p35 mRNA levels compared to the lipids of virulent S2P strain (LV). Neither LA nor LV induced anti-inflammatory IL-10. Regarding signalling pathways, we here report that LA induced a significant phosphorylation of p38 and extracellular signal-regulated kinases 1 and 2 (ERK1/2) whereas LV only induced a reduced activation of ERK1/2. Besides, NF-κB was activated by LA and LV, but LA produced an early degradation of the inhibitor IκB. Interestingly, LV and the majority of its lipid fractions, exerted a significant inhibition of concanavalin A-induced peripheral blood mononuclear cell proliferation with respect to LA and its corresponding lipid fractions. In addition, we determined that animals infected with R1A developed a higher increase in IgM anti-phosphatidylcholine than those inoculated with S2P. Collectively, S2P lipids generated a decreased inflammatory response contributing to the evasion of innate immunity. Moreover, since R1A lipids induced a pro-inflammatory profile, we propose these molecules as good candidates for immunoprophylactic strategies against babesiosis.

Babesia bovis biological clones and the inter-strain allelic diversity of the Bv80 gene support subpopulation selection as a mechanism involved in the attenuation of two virulent isolates.

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.

Acute and persistent infection by a transfected Mo7 strain of Babesia bovis.

A Mo7-derived Babesia bovis line stably transfected with the gfp-bsd gene was inoculated into two four to five months old calves, while two additional calves were inoculated with Mo7 parasites. Similar mild clinical signs were detected in all calves. B. bovis rap-1 was identified in the bloodstream by PCR four days post inoculation (dpi), and consistently over ten months thereafter. Transfusion of blood from experimentally infected calves into four naïve splenectomized calves at 212 dpi resulted in acute disease in recipients, confirming persistent infection in the four donor animals. The proportion of GFP expressing parasites recovered from a splenectomized recipient calf is undistinguishable from transfected parasites that were maintained in long term culture under blasticidin selection. Furthermore, the sequences of transfected genes in recovered parasites remained unaltered. Together, the data demonstrates that exogenous B. bovis transgenes can be expressed and remain stable throughout acute and persistent infection in calves.

Genetic diversity of Babesia bovis in virulent and attenuated strains.

The aim of this study was to compare the genetic diversity of the single copy Bv80 gene sequences of Babesia bovis in populations of attenuated and virulent parasites. PCR/ RT-PCR followed by cloning and sequence analyses of 4 attenuated and 4 virulent strains were performed. Multiple fragments in the range of 420 to 744 bp were amplified by PCR or RT-PCR. Cloning of the PCR fragments and sequence analyses revealed the presence of mixed subpopulations in either virulent or attenuated parasites with a total of 19 variants with 12 different sequences that differed in number and type of tandem repeats. High levels of intra- and inter-strain diversity of the Bv80 gene, with the presence of mixed populations of parasites were found in both the virulent field isolates and the attenuated vaccine strains. In addition, during the attenuation process, sequence analyses showed changes in the pattern of the parasite subpopulations. Despite high polymorphism found by sequence analyses, the patterns observed and the number of repeats, order, or motifs found could not discriminate between virulent field isolates and attenuated vaccine strains of the parasite.

Attenuation of virulence in an apicomplexan hemoparasite results in reduced genome diversity at the population level.

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.

Genome-wide analysis of peptidase content and expression in a virulent and attenuated Babesia bovis strain pair.

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.