Isolation of viable Babesia bovis merozoites to study parasite invasion.

Babesia parasite invades exclusively red blood cell (RBC) in mammalian host and induces alterations to host cell for survival. Despite the importance of Babesia in livestock industry and emerging cases in humans, their basic biology is hampered by lack of suitable biological tools. In this study, we aimed to develop a synchronization method for Babesia bovis which causes the most pathogenic form of bovine babesiosis. Initially, we used compound 2 (C2), a specific inhibitor of cyclic GMP-dependent protein kinase (PKG), and a derivative of C2, ML10. While both inhibitors were able to prevent B. bovis egress from RBC and increased percentage of binary forms, removal of inhibitors from culture did not result in a synchronized egress of parasites. Because using PKG inhibitors alone was not efficient to induce a synchronized culture, we isolated viable and invasive B. bovis merozoites and showed dynamics of merozoite invasion and development in RBCs. Using isolated merozoites we showed that BbVEAP, VESA1-export associated protein, is essential for parasite development in the RBC while has no significant role in invasion. Given the importance of invasion for the establishment of infection, this study paves the way for finding novel antigens to be used in control strategies against bovine babesiosis.

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.

Risks of cattle babesiosis (Babesia bovis) outbreaks in a semi-arid region of Argentina.

The epidemiology of Babesia bovis was studied in terms of enzootic stability/instability and husbandry and abiotic factors influencing B. bovis transmission rate in northeastern Santiago del Estero province, Argentina. The area is of limited suitability for its only vector in Argentina, the tick Rhipicephalus microplus. The proportion of calf herds in a state of enzootic stability/instability to B. bovis was determined and husbandry practices and abiotic factors associated with variations in B. bovis transmission rates were explored using a cross-sectional observational study design. Daily probability of infection (inoculation rate, h) with B. bovis was calculated from age-specific seroprevalence via ELISAi in 58 herds of 4.5-8.5-month-old calves. Herds were considered to be in enzootic instability (EI) when h < 0.005, and therefore inferred to be at risk of babesiosis outbreaks. Husbandry practices associated with differences in B. bovis transmission were analyzed using generalized linear models. Sixty-two percent of herds were found to be in an EI situation for B. bovis. Calves raised exclusively on permanent pastures -where higher cattle density is achieved- were exposed to higher B. bovis inoculation rates (h = 0.0063, 95% CI 0.0032-0.0123) than those reared under forage combinations (h = 0.0024, 95% CI 0.0011-0.0051) (P =  0.05). In addition, calves from herds located in the area of intermediate suitability for R. microplus development were more likely to become infected with B. bovis (h = 0.0067, 95% CI 0.0037-0.0121) than those reared in the ecologically unfavorable area for the vector (h = 0.0023, 95% CI 0.0010-0.0049) (P =  0.02). Neither the frequency of treatment with acaricides nor the use of long-acting acaricides to control R. microplus influenced the inoculation rate (P =  0.99 and P =  0.26, respectively). This result indicates that current R. microplus control schemes are not effective in reducing B. bovis transmission. Enzootic instability still prevails in the study area despite the drastic changes occurred in cattle production system. However, 38% of herds did reach enzootic stability; therefore, a specific epidemiological status cannot be assumed at a regional level. Yearly determination of the immunological status of each calf cohort is considered a proper approach to decision-making in vaccination against B. bovis.

Silencing expression of the Rhipicephalus microplus vitellogenin receptor gene blocks Babesia bovis transmission and interferes with oocyte maturation.

BACKGROUND: Rhipicephalus microplus is an efficient biological vector of Babesia bovis, a causative agent of bovine babesiosis. Babesia bovis is passed transovarially to the next generation of ticks, which then transmit the parasite to naïve animals. Due to the importance of the R. microplus ovary for tick reproduction and transmission of B. bovis, we investigated the hypothesis that silencing vitellogenin receptor gene expression in the ovary during tick feeding on B. bovis-infected cattle would affect parasite transmission to the next generation of ticks. RESULTS: Silencing expression of the vitellogenin receptor in the ovary by RNA interference, resulted in reduced tick fertility. We observed reduced egg production (i.e. reduced weight of eggs), a lower rate of embryonic development, and a reduction in hatching. Analysis of individual larvae by PCR confirmed that RNAi mediated downregulation of the R. microplus vitellogenin receptor and also interfered with transovarial transmission of B. bovis. None of the larvae (0/58) from the RmVgR dsRNA-injected group were PCR-positive, whereas 12% (7/58) and 17% (10/58) of larvae from the non-injected and buffer-injected control groups, respectively, were infected with B. bovis. CONCLUSIONS: The combined effects of reduced fecundity and reduced infection in surviving larvae resulting from silencing indicate that vitellogenin receptor is essential for tick reproduction and may play a vital role in B. bovis transmission.

Up-regulated expression of spherical body protein 2 truncated copy 11 in Babesia bovis is associated with reduced cytoadhesion to vascular endothelial cells.

The factors involved in gain or loss of virulence in Babesia bovis are unknown. Spherical body protein 2 truncated copy 11 (sbp2t11) transcripts in B. bovis were recently reported to be a marker of attenuation for B. bovis strains. Increased cytoadhesion of B. bovis-infected red blood cells (iRBC) to vascular endothelial cells is associated with severe disease outcomes and an indicator of parasite virulence. Here, we created a stable B. bovis transfected line over-expressing sbp2t11 to determine whether up-regulation of sbp2t11 is associated with changes in cytoadhesion. This line was designated sbp2t11up and five B. bovis clonal lines were derived from the sbp2t11up line by limiting dilution for characterisation. We compared the ability of iRBCs from the sbp2t11up line and its five derivative clonal lines to adhere to bovine brain endothelial cells, using an in vitro cytoadhesion assay. The same lines were selected for in vitro cytoadhesion and the levels of sbp2t11 transcripts in each selected line were quantified. Our results demonstrate that up-regulation of sbp2t11 is accompanied by a statistically significant reduction in cytoadhesion. Confirmed up-regulation of sbp2t11 in B. bovis concomitant with the reduction of iRBC in vitro cytoadhesion to bovine brain endothelial cell is consistent with our previous finding that up-regulation of sbp2t11 is an attenuation marker in B. bovis and suggests the involvement of sbp2t11 transcription in B. bovis virulence.

Retrospective study of epidemiological, clinical and pathological findings of bovine babesiosis in Mato Grosso do Sul, Brazil (1995 -2017).

Babesiosis is a tick-borne hemolytic disease caused by intraerythrocytic protozoan parasites of the genus Babesia. The present study describes epidemiological, clinical, and pathological features of cases of bovine babesiosis diagnosed over the last two decades in the state of Mato Grosso do Sul in central-western Brazil. Examination of a total of 5298 bovine samples resulted in a definitive or presumptive diagnosis of babesiosis in 41 cases from 33 outbreaks. Babesia bovis was the most common causal agent of bovine babesiosis, and animals of all ages were affected. Zebu cattle, which are more frequent in the study region than are taurine cattle, were the cattle most often affected. Although zebu cattle have been claimed to be highly tolerant, the present findings show that they can develop severe illness and die due to Babesia spp. infection. The disease was diagnosed almost every year over the time period covered in this study (1995-2017). However, the number of cases of bovine babesiosis diagnosed was low compared to the total number of cases reviewed. Veterinary practitioners that work on cattle farms should to be able to diagnose the disease in its early stages so that treatment can be initiated promptly and economic losses can be minimized.

Comparisons of the topographic characteristics and electrical charge distributions among Babesia-infected erythrocytes and extraerythrocytic merozoites using AFM.

Tick-borne Babesia parasites are responsible for costly diseases worldwide. Improved control and prevention tools are urgently needed, but development of such tools is limited by numerous gaps in knowledge of the parasite-host relationships. We hereby used atomic force microscopy (AFM) and frequency-modulated Kelvin probe potential microscopy (FM-KPFM) techniques to compare size, texture, roughness and surface potential of normal and infected Babesia bovis, B. bigemina and B. caballi erythrocytes to better understand the physical properties of these parasites. In addition, AFM and FM-KPFM allowed a detailed view of extraerythrocytic merozoites revealing shape, topography and surface potential of paired and single parasites. B. bovis-infected erythrocytes display distinct surface texture and overall roughness compared to noninfected erythrocytes. Interestingly, B. caballi-infected erythrocytes do not display the surface ridges typical in B. bovis parasites. Observations of extraerythrocytic B. bovis, B. bigemina and B. caballi merozoites using AFM revealed differences in size and shape between these three parasites. Finally, similar to what was previously observed for Plasmodium-infected erythrocytes, FM-KPFM images reveal an unequal electric charge distribution, with higher surface potential above the erythrocyte regions that are likely associated with Babesia parasites than over its remainder regions. In addition, the surface potential of paired extraerythrocytic B. bovis Mo7 merozoites revealed an asymmetric potential distribution. These observations may be important to better understand the unique cytoadhesive properties of B. bovis-infected erythrocytes, and to speculate on the role of differences in the distribution of surface charges in the biology of the parasites.

The Babesia bovis hap2 gene is not required for blood stage replication, but expressed upon in vitro sexual stage induction.

Babesia bovis, is a tick borne apicomplexan parasite responsible for important cattle losses globally. Babesia parasites have a complex life cycle including asexual replication in the mammalian host and sexual reproduction in the tick vector. Novel control strategies aimed at limiting transmission of the parasite are needed, but transmission blocking vaccine candidates remain undefined. Expression of HAP2 has been recognized as critical for the fertilization of parasites in the Babesia-related Plasmodium, and is a leading candidate for a transmission blocking vaccine against malaria. Hereby we identified the B. bovis hap2 gene and demonstrated that it is widely conserved and differentially transcribed during development within the tick midgut, but not by blood stage parasites. The hap2 gene was disrupted by transfecting B. bovis with a plasmid containing the flanking regions of the hap2 gene and the GPF-BSD gene under the control of the ef-1α-B promoter. Comparison of in vitro growth between a hap2-KO B. bovis clonal line and its parental wild type strain showed that HAP2 is not required for the development of B. bovis in erythrocytes. However, xanthurenic acid-in vitro induction experiments of sexual stages of parasites recovered after tick transmission resulted in surface expression of HAP2 exclusively in sexual stage induced parasites. In addition, hap2-KO parasites were not able to develop such sexual stages as defined both by morphology and by expression of the B. bovis sexual marker genes 6-Cys A and B. Together, the data strongly suggests that tick midgut stage differential expression of hap2 is associated with the development of B. bovis sexual forms. Overall these studies are consistent with a role of HAP2 in tick stages of the parasite and suggest that HAP2 is a potential candidate for a transmission blocking vaccine against bovine babesiosis.

Chemotherapeutic efficacies of a clofazimine and diminazene aceturate combination against piroplasm parasites and their AT-rich DNA-binding activity on Babesia bovis.

Recently, we reported that clofazimine (CF) has an anti-piroplasm activity, but it could not completely eliminate parasites in the host. The currently available anti-piroplasm drug, diminazene aceturate (DA), has sometimes been reported to have toxic side effects. In the present study, we evaluated the combination treatment with CF and DA against piroplasms both in vitro and in vivo. Additionally, mRNA level and DNA amounts were analyzed in CF‒ and DA‒treated Babesia bovis by a qPCR. The CF-DA combination had additive effects on Babesia bovis, B. bigemina, and B. caballi and synergistic effects on Theileria equi. The CF-DA combination chemotherapies against B. microti in mice were more potent than their monotherapies. In the CF‒ and DA‒treated B. bovis, CF dose-dependently down-regulated mRNA level and DNA amounts of extranuclear genes (AT-rich featured), whereas DA down-regulated only DNA amounts of extranuclear genes, but those of nuclear genes were slightly down- or up-regulated by CF and DA. In conclusion, the CF-DA combination has a higher efficiency against piroplasms than CF or DA monotherapies. CF and DA might have an AT-rich DNA-binding activity. All results suggest that the CF-DA combination chemotherapy will be a better choice to treat piroplasmosis instead of DA monotherapy.

In vitro culture of Babesia bovis in a bovine serum-free culture medium supplemented with insulin, transferrin, and selenite.

Bovine serum is an important factor for the optimal growth of Babesia bovis in vitro. This protozoan can be cultured in M-199 with Earle's salts medium (M-199) supplemented with 40% bovine serum (BS). In the present study, four media were assessed along with the control medium M-199. The effect on the proliferation of B. bovis in vitro was tested when these media were combined with insulin (Ins), transferrin (Trans) and selenite (Sel) in the absence of bovine serum. Treatment with Advanced DMEM/F12 medium (A-DMEM/F12) achieved the highest percentage of parasitized erythrocytes (PPE), reaching a maximum value of 9.59%. A-DMEM/F12 medium supplemented with a mixture of Ins (2000 mg/L), Trans (1100 mg/L), and Sel (1.34 mg/L) allowed for the adaptation and proliferation of B. bovis without bovine serum, showed a constant increase in PPE, and reached a maximum value of 9.7% during seven cycles of in vitro culture. It was concluded that continuous proliferation of B. bovis in vitro could be achieved using A-DMEM/F12 medium supplemented with Ins-Trans-Sel, without bovine serum. After adaptation for proliferation in serum-free medium, the B. bovis strain of parasites could have future use in the study of this economically important protozoan species that affects cattle.

RBC invasion and invasion-inhibition assays using free merozoites isolated after cold treatment of Babesia bovis in vitro culture.

Babesia bovis is an apicomplexan hemoprotozoan that can invade bovine red blood cells (RBCs), where it multiplies asexually. RBC invasion assays using free viable merozoites are now routinely used to understand the invasion mechanism of B. bovis, and to evaluate the efficacy of chemicals and antibodies that potentially inhibit RBC invasion by the parasite. The application of high-voltage pulses (high-voltage electroporation), a commonly used method to isolate free merozoites from infected RBCs, reduces the viability of the merozoites. Recently, a cold treatment of B. bovis in vitro culture was found to induce an effective release of merozoites from the infected RBCs. In the present study, we incubated in vitro cultures of B. bovis in an ice bath to liberate merozoites from infected RBCs and then evaluated the isolated merozoites in RBC invasion and invasion-inhibitions assays. The viability of the purified merozoites (72.4%) was significantly higher than that of merozoites isolated with high-voltage electroporation (48.5%). The viable merozoites prepared with the cold treatment also invaded uninfected bovine RBCs at a higher rate (0.572%) than did merozoites prepared with high-voltage electroporation (0.251%). The invasion-blocking capacities of heparin, a polyclonal rabbit antibody directed against recombinant B. bovis rhoptry associated protein 1, and B. bovis-infected bovine serum were successfully demonstrated in an RBC invasion assay with the live merozoites prepared with the cold treatment, suggesting that the targets of these inhibitors were intact in the merozoites. These findings indicate that the cold treatment technique is a useful tool for the isolation of free, viable, invasion-competent B. bovis merozoites, which can be effectively used for RBC invasion and invasion-inhibition assays in Babesia research.

Molecular and serological detection of Babesia bovis- and Babesia bigemina-infection in bovines and water buffaloes raised jointly in an endemic field.

Babesia bovis and Babesia bigemina are causative agents of bovine babesiosis, a tick-borne disease of cattle in tropical and subtropical regions. Babesia spp. infection adversely affects cattle health and can be fatal resulting in considerable economic loss worldwide. Under endemic stability conditions, herds contain high numbers of chronically infected, asymptomatic carrier animals, in which no parasitemia is detected by microscopic blood smear examination. In addition to bovines, also water buffaloes are infected by both Babesia spp. commonly leading to a subclinical infection. The infection rate (by nPCR) and herd exposure (by IFAT) of bovines and water buffaloes reared under similar field conditions in an area of endemic stability were determined and compared. In order to optimize direct parasite detection, highly sensitive nPCR assays were developed and applied, allowing the detection of as little as 0.1 fg DNA of each Babesia pathogen. Significantly lower percentages (p<0.001) of seropositive water buffaloes compared to bovines were observed for B. bovis (71.4% vs. 98%) and B. bigemina (85% vs. 100%). Interestingly, in comparison, differences noticed between water buffaloes and bovines were considerably larger with direct parasite detection by nPCR (16.2% vs. 82.3% and 24% vs. 94.1% for B. bovis and B. bigemina, respectively). As expected, bovines subjected to monthly acaricide applications exhibited a significant lower infection rate as determined by nPCR than bovines not subjected to these measures (B. bovis 33.3% vs. 90.7%, p<0.001; B. bigemina 80% vs. 96.5%, p<0.001, for treated vs. untreated animals). Interestingly no differences between these groups were observed with respect to seropositivity, suggesting similar rates of parasite exposure (B. bovis 100% vs. 97.7%, p<0.001; B. bigemina 100% vs. 100%, p<0.001). Importantly, a significantly higher number of water buffaloes as determined by nPCR were infected when reared jointly with bovines not subjected to tick control than when reared jointly with bovines subjected to tick control (B. bovis 31.6% vs. 9.5%, p<0.01; B. bigemina 42.1% vs. 9.5%, p<0.01, for water buffaloes reared with untreated vs. treated bovines) and/or when reared without bovines (B. bovis 31.6% vs. 11.6%, p<0.01; B. bigemina 42.1% vs. 20%, p<0.01). An accumulation of seropositivity and a decline of infection rates were observed in older animals, while differences observed with regard to gender may warrant further investigation. In summary, our findings suggest that water buffaloes are much more capable to limit or eliminate Babesia infection, possibly due to a more capable immune defense. Furthermore, an increased Babesia spp. parasite reservoir of bovines seems to increase the infection rate of water buffaloes when both are reared on the same pasture.

Shared elements of host-targeting pathways among apicomplexan parasites of differing lifestyles.

Apicomplexans are a diverse group of obligate parasites occupying different intracellular niches that require modification to meet the needs of the parasite. To efficiently manipulate their environment, apicomplexans translocate numerous parasite proteins into the host cell. Whereas some parasites remain contained within a parasitophorous vacuole membrane (PVM) throughout their developmental cycle, others do not, a difference that affects the machinery needed for protein export. A signal-mediated pathway for protein export into the host cell has been characterized in Plasmodium parasites, which maintain the PVM. Here, we functionally demonstrate an analogous host-targeting pathway involving organellar staging prior to secretion in the related bovine parasite, Babesia bovis, a parasite that destroys the PVM shortly after invasion. Taking into account recent identification of a similar signal-mediated pathway in the coccidian parasite Toxoplasma gondii, we suggest a model in which this conserved pathway has evolved in multiple steps from signal-mediated trafficking to specific secretory organelles for controlled secretion to a complex protein translocation process across the PVM.

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.

Calcium ions are involved in egress of Babesia bovis merozoites from bovine erythrocytes.

Bovine babesiosis is a livestock disease known to cause economic losses in endemic areas. The apicomplexan parasite Babesia bovis is able to invade and destroy the host's erythrocytes leading to the serious pathologies of the disease, such as anemia and hemoglobinuria. Understanding the egress mechanisms of this parasite is therefore a key step to develop new therapeutic strategies. In this study, the possible involvement of Ca(2+) in the egress of B. bovis merozoites from infected erythrocytes was investigated. Egress was artificially induced in vitro using calcium ionophore A23187 and thapsigargin to increase Ca(2+) concentration in the cytosol of the parasite cells. The increased intracellular Ca(2+) concentration following these treatments was confirmed using live cell Ca(2+) imaging with confocal laser scanning microscopy. Based on our findings, we suggest a Ca(2+) signalling pathway in the egress of B. bovis merozoites.

The glycosylphosphatidylinositol-anchored protein repertoire of Babesia bovis and its significance for erythrocyte invasion.

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.

Specific antibody to a conserved region of Babesia apical membrane antigen-1 inhibited the invasion of B. bovis into the erythrocyte.

Apical membrane antigen-1 (AMA-1) is a microneme protein that exists in all apicomplexan parasites and plays an indispensable role in the invasion into host cell. Central region of ectodomains I and II of Babesia bovis apical membrane antigen-1 (BbAMA-1P) is highly conserved with these of Babesia species and may be beneficial for vaccine development against babesiosis. In the present study, recombinant protein encoding the central region of B. bovis AMA-1 (rBbAMA-1P) was produced in Escherichia coli and its antiserum was prepared in mice for further molecular characterization. Anti-rBbAMA-1P serum specifically reacted with corresponding authentic protein of B. bovis as determined by Western blotting and IFAT. Cultured B. bovis treated with anti-rBbAMA-1P serum showed significant reduction in the in vitro growth of the parasites. Moreover, preincubated free merozoites with 1mg/ml anti-rBbAMA-1P serum inhibited their efficiency in the invasion into erythrocytes (RBCs) by 61% and 70% at 3h and 6h, respectively. Our data suggest that the central region of domains I and II of BbAMA-1 may serve as a vaccine candidate against babesiosis.

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.

Gut transcriptome of replete adult female cattle ticks, Rhipicephalus (Boophilus) microplus, feeding upon a Babesia bovis-infected bovine host.

As it feeds upon cattle, Rhipicephalus (Boophilus) microplus is capable of transmitting a number of pathogenic organisms, including the apicomplexan hemoparasite Babesia bovis, a causative agent of bovine babesiosis. The R. microplus female gut transcriptome was studied for two cohorts: adult females feeding on a bovine host infected with B. bovis and adult females feeding on an uninfected bovine. RNA was purified and used to generate a subtracted cDNA library from B. bovis-infected female gut, and 4,077 expressed sequence tags (ESTs) were sequenced. Gene expression was also measured by a microarray designed from the publicly available R. microplus gene index: BmiGI Version 2. We compared gene expression in the tick gut from females feeding upon an uninfected bovine to gene expression in tick gut from females feeding upon a splenectomized bovine infected with B. bovis. Thirty-three ESTs represented on the microarray were expressed at a higher level in female gut samples from the ticks feeding upon a B. bovis-infected calf compared to expression levels in female gut samples from ticks feeding on an uninfected calf. Forty-three transcripts were expressed at a lower level in the ticks feeding upon B. bovis-infected female guts compared with expression in female gut samples from ticks feeding on the uninfected calf. These array data were used as initial characterization of gene expression associated with the infection of R. microplus by B. bovis.

Bovine babesiosis in the 21st century: advances in biology and functional genomics.

Bovine babesiosis caused by the protozoan parasite, Babesia bovis, remains a significant cause of avoidable economic losses to the livestock industry in many countries throughout the world. The molecular mechanisms underlying the pathophysiology of severe disease in susceptible cattle are not well understood and the tools available to study the biology of the parasite, including technologies for genetic manipulation, have only recently been developed. Recent availability of multiple parasite genomes and bioinformatic tools, in combination with the development of new biological reagents, will facilitate our better understanding of the parasite. This will ultimately assist in the identification of novel targets for the development of new therapeutics and vaccines. Here we describe some recent advances in Babesia research and highlight some important challenges for the future.