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The tick-borne apicomplexan parasite Babesia bovis causes bovine babesiosis which leads to enormous food and economic losses around the world. The existing resources to manage this disease are limited and have pitfalls, therefore, introduction of new strategies is urgently needed. B. bovis reproduces sexually in the midgut of its tick vector. HAP2, a well conserved ancient protein, plays a crucial role in the gamete fusion of this parasite and is a strong candidate for developing transmission-blocking vaccines. We previously demonstrated that immunization of cattle with full size B. bovis HAP2 blocks transmission of the parasite by Rhipicephalus microplus . Understanding the conserved structural features and antigenicity of HAP2 protein and its domains will facilitate developing effective methods to control pathogen transmission. In this study, we analyzed and compared AlphaFold2-predicted 3D structure of B. bovis HAP2 with the well-characterized crystal structures of HAP2 of Chlamydomonas reinhardtii and Arabidopsis thaliana . The comparisons and structural analysis resulted in the definition of three domains’ sequences, fusion loops, and disulfide bonds in the B. bovis HAP2. In addition, recombinant versions of each three predicted HAP2 domains were recognized by antibodies from HAP2 immunized and transmission-protected cattle, confirming their antigenicity. Remarkably, domain II was highly recognized compared to the other two domains. This study introduces new directions in designing novel functional assays and improved vaccine design through targeting the HAP2 protein.

Background Bovine babesiosis is a tick-borne disease that poses a significant economic threat to cattle industries in tropical and subtropical areas, and Babesia bovis is the most virulent causative agent of bovine babesiosis. This apicomplexan parasite infects erythrocytes of cattle, causing severe hemolytic disease, and animals that survive an acute infection become persistently infected for life. Adult cattle (> 1 year of age) are highly susceptible and often succumb to acute infection. Protective host immunity involves peripheral blood mononuclear cells (PBMCs) including monocytes, dendritic cells (DC), natural killer (NK), T cells, and B cells, all of which act to control the pathogen. Monocytes release the cytokines interleukin (IL)-1β and tumor necrosis factor (TNF) and nitric oxide, in addition to chemokines that attract immature DCs. NK cells release IL-12, IL-18, and interferon gamma (IFNγ). Mature DC migrate to secondary lymphoid tissues to present Babesia antigens to T cells. B cells will produce antibodies against Babesia . Methods In this study, we examined the transcriptional signatures of PBMCs from adult cattle (aged > 1.5 years) experimentally infected with the B. bovis virulent strain Vir-S74-T3Bo, during the acute phase of babesiosis, at 10 days post infection (dpi), using RNA Sequencing (RNA-Seq) technology. Results Transcriptional signatures evident during the acute phase of babesiosis were cytokines and chemokines, such as IL-0, TNF, IL-1B, IL-18, CSF1, CXCL10 and CXCL16; pattern recognition receptors, such as CD14, TLR and NOD2; complement components, such as C1R, C2, C3aR1, CFB, CFI and CFP; cell adhesion molecules, such as ICAM1/2 and SELL; and apoptosis markers, such as CASP, BAX and BAK. We identified 1766 upregulated and 1508 downregulated genes, with fold changes ranging from two- to 429-fold. We discuss our findings in the context of immune responses to acute disease as a mechanism for adult host survival, with a focus on the molecular functions and biological processes involved in the response to B. bovis infection. Conclusions In this RNA-Seq analysis, we identified genes that are up- and downregulated in response to acute B. bovis infection. Gene expression of IL-10, along with that of the inflammatory cytokines IL-1β, TNFα and IL-18, suggests a non-protective response to B. bovis at 10 dpi. These results enhance our understanding of the molecular interactions between Babesia and the host immune system. Graphical Abstract

The apicomplexan tickborne parasites Babesia bovis and B. bigemina are the major causative agents of bovine babesiosis, a disease that negatively affects the cattle industry and food safety around the world. The absence of correlates of protection represents one major impediment for the development of effective and sustainable vaccines against bovine babesiosis. Herein we superinfected cattle with attenuated and virulent strains of B. bovis to investigate immune correlates of protection against acute bovine babesiosis. Three 6-month-old Holstein calves were infected intravenously (IV) with the in vitro culture attenuated Att-S74-T3Bo B. bovis strain (10 6 infected bovine red blood cells (iRBC)/calf) while three age-matched Holstein calves were inoculated IV with normal RBC as controls (10 6 RBC/calf). All Att-S74-T3Bo-infected calves showed a significant increase in temperature early after inoculation but recovered without treatment. Att-S74-T3Bo-infected calves also developed: (a) monocytosis, neutropenia, and CD4 + lymphopenia in peripheral blood on days 3 to 7 post-inoculation; (b) significant levels of TNFα, CXCL10, IFNγ, IL-4, and IL-10 in sera at day 6 after infection; and (c) IgM and IgG against B. bovis antigens, starting at days 10 and 30 post-inoculation, respectively. At 46 days post-Att-S74-T3Bo inoculation, all experimental calves were infected IV with the homologous virulent B. bovis strain Vir-S74-T3Bo (10 7 iRBC/calf). All Att-S74-T3Bo-infected calves survived superinfection with Vir-S74-T3Bo without displaying signs of acute babesiosis. In contrast, control animals showed signs of acute disease, starting at day 10 post-Vir-S74-T3Bo infection, and two of them were humanely euthanized at days 13 and 14 after inoculation due to the severity of their symptoms. Also, control calves showed higher (P<0.05) parasite load in peripheral blood compared to animals previously exposed to Att-S74-T3Bo. No significant alterations in the profile of leukocytes and cytokines were observed in Att-S74-T3Bo-inoculated after Vir-S74-T3Bo infection. In conclusion, data demonstrate novel changes in the profile of blood immune cells and cytokine expression in peripheral blood that are associated with protection against acute bovine babesiosis. These identified immune correlates of protection may be useful for designing effective and sustainable vaccines against babesiosis in cattle.

IntroductionLive in vivo attenuated Babesia bovis vaccines produced by sequential passages in splenectomized calves have historically been used to control acute bovine babesiosis in endemic areas worldwide. However, several constraints prevent the widespread use of these vaccines, including the need for several splenectomized calves to produce vaccine batches, and potential inconsistent parasite attenuation, which contraindicates their use for highly Babesia -susceptible adult cattle. Thus, the use of vaccines based on well-defined in vitro culture attenuated B. bovis strains emerges as a more sustainable and efficient alternative. Previous work demonstrated that the culture attenuated strain Att-S74-T3Bo is non-tick transmissible and able to safely protect calves against needle challenge with a B. bovis virulent strain.Methods and resultsHerein we evaluated safety and efficacy of Att-S74-T3Bo in preventing acute babesiosis in adult (>1.5 year of age) cattle. Results demonstrated that Att-S74-T3Bo vaccination of adult animals (n=5) induced self-limiting signs of acute infection and protected the vaccinated animals against challenge with the homologous virulent B. bovis strain Vir-S74-T3Bo. Att-S74-T3Bo-vaccinated adult cattle developed significant (P<0.05) monocytosis, with concomitant neutropenia and CD4+ leukopenia, in peripheral blood early after vaccination. Also, vaccinated animals developed a specific signature of pro- and anti-inflammatory cytokine expression in peripheral blood and significant levels of IgM, total IgG, IgG1, and IgG2 against the B. bovis immunodominant antigen RAP-1 CT. Strikingly, none of the vaccinated animals showed any signs of acute babesiosis after challenge with Vir-S74-T3Bo. In contrast, control adult cattle (n=5) showed pathognomonic symptoms of acute babesiosis, and significant decrease (P<0.05) in lymphocytes, monocytes, and neutrophils, starting on day 7 post-challenge. All control animals developed severe acute disease and were euthanized on days 10 through 12 days post-challenge.Discussion and conclusionEvidence from this study indicates that Att-S74-T3Bo safely protects highly susceptible adult cattle against challenge with a homologous virulent strain of B. bovis . In conclusion, Att-S74-T3Bo may be considered as a potential efficient and sustainable attenuated candidate vaccine strain to control acute bovine babesiosis in highly susceptible adult cattle. Future studies should focus on increasing the number of animals vaccinated, duration of immunity, and efficacy of this attenuated strain against heterologous virulent parasite strains.

, 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 , 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. Sequence comparisons among the RRA sequences of several strains and other 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 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. 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 RRA, which is recognized by sera from protected bovines, supports its inclusion in future subunit epitope-based vaccines against .

Babesia bigemina is a tick-borne apicomplexan hemoprotozoan responsible for bovine babesiosis. The current drugs used for bovine babesiosis treatment have several drawbacks, including toxicity, the lack of effectiveness to clear the parasite, and potential to develop resistance. Identifying compounds that target essential and unique parasite metabolic pathways is a rational approach toward finding alternative drug treatments. Based on the genome sequence and transcriptomics analysis, it can be inferred that anaerobic glycolysis is the dominant adenosine triphosphate (ATP) supply for Babesia , and lactate dehydrogenase (LDH) is one of the essential enzymes in this pathway. Furthermore, the Babesia LDH sequence is distinct from its bovine homologue and thus a potential chemotherapeutic target that would result in decreasing the ATP supply to the parasite but not to the host. Gossypol is a known efficient specific inhibitor of LDH in the sensu stricto B. bovis and the sensu lato B. microti , among other related parasites, but no such data are currently available in the sensu stricto B. bigemina parasites. Hereby, we show that the LDH amino acid sequence is highly conserved among sensu stricto but not in sensu lato Babesia spp. A predictive structural analysis of B. bigemina LDH showed the conservation of the key amino acids involved in the binding to gossypol compared to B. bovis . Gossypol has a significant (P < 0.0001) inhibitory effect on the in vitro growth of B. bigemina , with IC 50 of 43.97 mM after 72 h of treatment. The maximum IC (IC 98 ) was observed at 60 mM gossypol. However, a significant effect on the viability of cattle PBMC was observed when the cells were cultured with 60 mM (IC 98 ) gossypol compared with DMSO-exposed control cells. Interestingly, B. bigemina cultured at 3% oxygen expresses significantly higher levels of LDH and is more resistant to gossypol than the parasites maintained at ambient conditions containing ~20% oxygen. Altogether, the results suggest the potential of gossypol as an effective drug against B. bigemina infection, but the risk of host toxicity at therapeutic doses should be further evaluated in in vivo studies.

Babesiosis is an acute and persistent tick-borne disease caused by protozoan parasites of the genus Babesia . These hemoparasites affect vertebrates globally, resulting in symptoms such as high fever, anemia, jaundice, and even death. Advancements in molecular parasitology revealed new Babesia species/genotypes affecting sheep and goats, including Babesia aktasi n. sp., which is highly prevalent in goats from Turkiye’s Mediterranean region. The objective of this study was to investigate the pathogenesis of B. aktasi infection in immunosuppressed (n=7) and non-immunosuppressed (n=6) goats. These animals were experimentally infected with fresh B. aktasi infected blood, and their clinical signs, hematological and serum biochemical parameters were monitored throughout the infection. The presence of parasites in the blood of immunosuppressed goats was detected by microscopic examination between 4 and 6 days after infection, accompanied by fever and increasing parasitemia. Goats that succumbed acute disease exhibited severe clinical signs, such as anemia, hemoglobinuria, and loss of appetite. However, the goats that survived showed milder clinical signs. In the non-immunosuppressed group, piroplasm forms of B. aktasi were observed in the blood within 2-5 days after inoculation, but with low (0.01-0.2%) parasitemia. Although these goats showed loss of appetite, typical signs of babesiosis were absent except for increased body temperature. Hematological analysis revealed significant decreases in the levels of red blood cells, leukocytes and platelet values post-infection in immunosuppressed goats, while no significant hematological changes were observed in non-immunosuppressed goats. In addition, serum biochemical analysis showed elevated transaminase liver enzymes levels, decreased glucose, and lower total protein values in the immunosuppressed group post-infection. Babesia aktasi , caused mild disease with minor clinical symptoms in non-immunosuppressed goats. However, in immunosuppressed goats, it exhibited remarkable pathogenicity, leading to severe clinical infections and death. In conclusion, this study provides valuable insights into the pathogenicity of the parasite and will serve as a foundation for future research aimed at developing effective prevention and control strategies against babesiosis in small ruminants. Further research is required to investigate the pathogenicity of B. aktasi in various goat breeds, other potential hosts, the vector ticks involved, and its presence in natural reservoirs.

The Rhipicephalus microplus tick is a notorious blood-feeding ectoparasite of livestock, especially cattle, responsible for massive losses in animal production. It is the main vector for transmission of pathogenic bacteria and parasites, including Babesia bovis, an intraerythrocytic apicomplexan protozoan parasite responsible for bovine Babesiosis. This study describes the development and testing of a live B. bovis vaccine expressing the protective tick antigen glutathione-S-transferase from Haemaphysalis longicornis (HlGST). The B. bovis S74-T3B parasites were electroporated with a plasmid containing the bidirectional Ef-1α (elongation factor 1 alpha) promoter of B. bovis controlling expression of two independent genes, the selectable marker GFP-BSD (green fluorescent protein-blasticidin deaminase), and HlGST fused to the MSA-1 (merozoite surface antigen 1) signal peptide from B. bovis. Electroporation followed by blasticidin selection resulted in the emergence of a mixed B. bovis transfected line (termed HlGST) in in vitro cultures, containing parasites with distinct patterns of insertion of both exogenous genes, either in or outside the Ef-1α locus. A B. bovis clonal line termed HlGST-Cln expressing intracellular GFP and HlGST in the surface of merozoites was then derived from the mixed parasite line HlGST using a fluorescent activated cell sorter. Two independent calf immunization trials were performed via intravenous inoculation of the HlGST-Cln and a previously described control consisting of an irrelevant transfected clonal line of B. bovis designated GFP-Cln. The control GFP-Cln line contains a copy of the GFP-BSD gene inserted into the Ef-1α locus of B. bovis in an identical fashion as the HIGST-Cln parasites. All animals inoculated with the HlGST-Cln and GFP-Cln transfected parasites developed mild babesiosis. Tick egg fertility and fully engorged female tick weight was reduced significantly in R. microplus feeding on HlGST-Cln-immunized calves. Collectively, these data show the efficacy of a transfected HlGST-Cln B. bovis parasite to induce detectable anti-glutathione-S-transferase antibodies and a reduction in tick size and fecundity of R. microplus feeding in experimentally inoculated animals.

is an apicomplexan parasite responsible for bovine babesiosis, a tick-borne disease with a worldwide impact. The disease remains inefficiently controlled, and few effective drugs, including imidocarb dipropionate (ID), are currently available in endemic areas. The objective of this study was to evaluate whether buparvaquone (BPQ), a drug currently used to treat cattle infected with the -related parasites, could be active against parasites. Herein, we compared the effect of ID and BPQ on growth erythrocyte culture. We compared the effect of ID and BPQ on the culture-adapted Texas T2Bo strain of cultured parasites were incubated with ID and BPQ at two starting parasitemia levels (PPE), 0.2% and 1%. cultured parasites were treated with ID or BPQ at concentrations ranging from 10 to 300 nM, during 4 consecutive days. Parasitemia levels were daily evaluated using microscopic examination. Data was compared using the independent Student's t-test. Both ID and BPQ significantly inhibited ( < 0.05) the growth of , regardless of the initial parasitemia used. At 1% parasitemia, BPQ had lower calculated inhibitory concentration 50 (IC50: 50.01) values than ID (IC50: 117.3). No parasites were found in wells with 0.2% starting parasitemia, treated previously with 50 nM of BPQ or ID, after 2 days of culture without drugs. At 1% parasitemia, no parasite survival was detected at 150 nM of BPQ or 300 nM of ID, suggesting that both drugs acted as babesiacidals. Overall, the data suggests that BPQ is effective against and shows a residual effect that seems superior to ID, which is currently the first-line drug for treating bovine babesiosis globally.

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.