Biochemical analysis of Hyalomma dromedarii salivary glands and gut tissues using SR-FTIR micro-spectroscopy.

Ticks are obligatory voracious blood feeders infesting diverse vertebrate hosts, that have a crucial role in the transmission of diverse pathogens that threaten human and animal health. The continuous emergence of tick-borne diseases due to combined worldwide climatic changes, human activities, and acaricide-resistant tick strains, necessitates the development of novel ameliorative tick control strategies such as vaccines. The synchrotron-based Fourier transform infrared micro-spectroscopy (SR-FTIR) is a bioanalytical microprobe capable of exploring the molecular chemistry within microstructures at a cellular or subcellular level and is considered as a nondestructive analytical approach for biological specimens. In this study, SR-FTIR analysis was able to explore a qualitative and semi-quantitative biochemical composition of gut and salivary glands of Hyalomma dromedarii (H. dromedarii) tick detecting differences in the biochemical composition of both tissues. A notable observation regarding Amide I secondary structure protein profile was the higher ratio of aggregated strands in salivary gland and beta turns in gut tissues. Regarding the lipid profile, there was a higher intensity of lipid regions in gut tissue when compared to salivary glands. This detailed information on the biochemical compositions of tick tissues could assist in selecting vaccine and/or control candidates. Altogether, these findings confirmed SR-FTIR spectroscopy as a tool for detecting differences in the biochemical composition of H. dromedarii salivary glands and gut tissues. This approach could potentially be extended to the analysis of other ticks that are vectors of important diseases such as babesiosis and theileriosis.

Impact of Equine and Camel Piroplasmosis in Egypt: How Much Do We Know about the Current Situation?

Piroplasmosis is a global tick-borne disease caused by hemoprotozoan parasites, which causes high morbidity and substantial economic losses in farm animals. Equine and camel piroplasmosis causes significant losses worldwide and in Egypt. The multifactorial effects and overall impact of equine and camel piroplasmosis in Egypt remain poorly characterized. However, several Babesia and Theileria spp. as well as potential tick vectors affecting these two species have been identified in the country. Equine and camel piroplasmosis has been reported by all governates in the country. Thus, in this work, we intend to provide a broad depiction of the current approaches used for diagnosis and control and the impact of piroplasmosis on the equine and camel industries in Egypt. We also identified current gaps in knowledge that might help develop future research efforts towards improved intervention and control of equine and camel piroplasmosis. It is important to develop specific diagnostic tools suitable for the early and chronic diagnosis of this disease. Altogether, the current situation warrants the development of large-scale epidemiological studies in order to obtain an accurate estimate for equine and camel piroplasmosis to secure the highly needed food resources in the country.

Experimental infection of non-immunosuppressed and immunosuppressed goats reveals differential pathogenesis of Babesia aktasi n. sp.

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.

Vaccination of cattle with the Babesia bovis sexual-stage protein HAP2 abrogates parasite transmission by Rhipicephalus microplus ticks.

The apicomplexan parasite Babesia bovis is responsible for bovine babesiosis, a poorly controlled tick-borne disease of global impact. The widely conserved gametocyte protein HAPLESS2/GCS1 (HAP2) is uniquely expressed on the surface of B. bovis sexual stage parasites and is a candidate for transmission-blocking vaccines (TBV). Here, we tested whether vaccination of calves with recombinant HAP2 (rHAP2) interferes with the transmission of B. bovis by competent ticks. Calves vaccinated with rHAP2 (n = 3), but not control animals (n = 3) developed antibodies specific to the vaccine antigen. Vaccinated and control animals were infested with Rhipicephalus microplus larvae and subsequently infected with virulent blood stage B. bovis parasites by needle inoculation, with all animals developing clinical signs of acute babesiosis. Engorged female ticks fed on the infected calves were collected for oviposition, hatching, and obtention of larvae. Transmission feeding was then conducted using pools of larvae derived from ticks fed on rHAP2-vaccinated or control calves. Recipient calves (n = 3) exposed to larvae derived from control animals, but none of the recipient calves (n = 3) challenged with larvae from ticks fed on rHAP2-vaccinated animals, developed signs of acute babesiosis within 11 days after tick infestation. Antibodies against B. bovis antigens and parasite DNA were found in all control recipient animals, but not in any of the calves exposed to larvae derived from HAP2-vaccinated animals, consistent with the absence of B. bovis infection via tick transmission. Overall, our results are consistent with the abrogation of parasite tick transmission in rHAP2-vaccinated calves, confirming this antigen as a prime TBV candidate against B. bovis.

Cross-sectional analysis of Piroplasma species-infecting camel (Camelus dromedaries) in Egypt using a multipronged molecular diagnostic approach.

Camel piroplasmosis is a tick-borne disease (TBD) caused by hemoprotozoan parasites. Hereby, we describe a cross-sectional study aiming at identifying Piroplasma spp.-infecting camels in Egypt using a multipronged molecular diagnostic approach. A total of 531 blood samples from camels (Camelus dromedarius) were collected from slaughterhouses at different governorates in Egypt for analysis during the period from June 2018 to May 2019. Piroplasma spp. was identified using microscopical examination and several different and sequential polymerase chain reaction (PCR) assays targeting the 18S rRNA genes. The overall prevalence of Piroplasma spp. in microscopical and molecular analyses in the samples was 11% (58/531) and 38% (203/531), respectively. Further discriminative multiplex PCR analysis targeting the 18S rRNA gene applied on all Piroplasma spp.-positive samples allowed the detection of Theileria equi (41%), Babesia caballi (5.4%), Babesia bigemina (0.5%), and Babesia bovis (4%). Additionally, the blast analysis of nested (n) PCR, targeting the V4 region, amplicon sequences resulted in the identification of B. vulpes (22%), Babesia sp. (9%), and Theileria sp. (3%). Overall, the results of this study confirmed the high prevalence of TBDs caused by several types of piroplasm hemoparasites in camel and suggests the need for future interventions aimed at improving the control of these potentially debilitating diseases that may be t-hreatening important economic resources and food security in Egypt.

Identification of novel immune correlates of protection against acute bovine babesiosis by superinfecting cattle with in vitro culture attenuated and virulent Babesia bovis strains.

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 (106 infected bovine red blood cells (iRBC)/calf) while three age-matched Holstein calves were inoculated IV with normal RBC as controls (106 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 (107 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.

Identification of CCp5 and FNPA as Novel Non-canonical Members of the CCp Protein Family in Babesia bovis.

Bovine babesiosis, caused by Babesia bovis, is an economically significant tick-borne disease that imposes restrictions to livestock production worldwide. Current methods to control bovine babesiosis have severe limitations and novel approaches, including transmission-blocking vaccines, are needed. Members of the widely conserved CCp family are multidomain adhesion proteins containing LCCL motifs, which are differentially expressed on gametocytes of apicomplexans, including Babesia spp. and Plasmodium spp. While Plasmodium parasites contain 6 distinct CCp genes, only three members (CCp 1-3) were previously identified in B. bovis. In this study, we describe the identification and characterization of two novel non-canonical members of the CCp gene family in B. bovis, named CCp5 and FNPA. The genes were identified in silico by TBLASTN using P. falciparum CCp family domains as queries. Unlike CCp1-3, the B. bovis CCp5 and FNPA proteins lack the LCCL canonical domain but contain other typical multidomain adhesion motifs which are present in classical CCp proteins. In addition, the B. bovis CCp5 and FNPA are in synteny with known CCp genes in related apicomplexans. Sequence analysis of these two proteins demonstrated high sequence conservation among B. bovis different isolates. Transcription, immunoblot, and immunofluorescence analyses demonstrated expression of CCp5 and FNPA in blood and in vitro induced sexual stages of B. bovis. The FNPA, in contrast to CCp5, has a predicted transmembrane domain, suggesting that it might be expressed in the surface of sexual stage parasites. Altogether, finding of this study support FNPA as a possible target of a transmission-blocking vaccine against B. bovis.

Unraveling the Complexity of the Rhomboid Serine Protease 4 Family of Babesia bovis Using Bioinformatics and Experimental Studies.

Babesia bovis, a tick-transmitted apicomplexan protozoon, infects cattle in tropical and subtropical regions around the world. In the apicomplexans Toxoplasma gondii and Plasmodium falciparum, rhomboid serine protease 4 (ROM4) fulfills an essential role in host cell invasion. We thus investigated B. bovis ROM4 coding genes; their genomic organization; their expression in in vitro cultured asexual (AS) and sexual stages (SS); and strain polymorphisms. B. bovis contains five rom4 paralogous genes in chromosome 2, which we have named rom4.1, 4.2, 4.3, 4.4 and 4.5. There are moderate degrees of sequence identity between them, except for rom4.3 and 4.4, which are almost identical. RT-qPCR analysis showed that rom4.1 and rom4.3/4.4, respectively, display 18-fold and 218-fold significantly higher (p < 0.01) levels of transcription in SS than in AS, suggesting a role in gametogenesis-related processes. In contrast, transcription of rom4.4 and 4.5 differed non-significantly between the stages. ROM4 polymorphisms among geographic isolates were essentially restricted to the number of tandem repeats of a 29-amino acid sequence in ROM4.5. This sequence repeat is highly conserved and predicted as antigenic. B. bovis ROMs likely participate in relevant host−pathogen interactions and are possibly useful targets for the development of new control strategies against this pathogen.

A Culture-Adapted Strain of Babesia bovis Has Reduced Subpopulation Complexity and Is Unable to Complete Its Natural Life Cycle in Ticks.

Babesia bovis natural field strains are composed of several geno-phenotypically distinct subpopulations. This feature, together with possible epigenetic modifications, may facilitate adaptation to variable environmental conditions. In this study we compare geno-phenotypical features among long-term (more than 12 years) (LTCP) and short-term cultured B. bovis parasites (STCP) derived from the B. bovis S74-T3Bo strain. LTCPs intraerythrocytic forms are smaller in size than STCPs and have faster in vitro growth rate. In contrast to its parental strain, the LTCP lack expression of the sexual stage specific 6cysA and 6cysB proteins and are unable to develop sexual forms upon in vitro sexual stage induction. Consistently, in contrast to its parental strain, LTCPs have reduced virulence and are not transmissible to cattle by vector competent Rhipicephalus microplus (R. microplus). Similar to previous comparisons among attenuated and virulent B. bovis strains, the LTCP line has decreased genomic diversity compared to the STCP line. Thus, LTCP may contribute to our understanding of adaptive mechanisms used by the parasites in response to environmental changes, protective immunity, virulence, and transmission by ticks. In addition, LTCPs may be considered as candidates for a non-tick transmissible vaccine against bovine babesiosis.

Harnessing Mycobacterium bovis BCG Trained Immunity to Control Human and Bovine Babesiosis.

Babesiosis is a disease caused by tickborne hemoprotozoan apicomplexan parasites of the genus Babesia that negatively impacts public health and food security worldwide. Development of effective and sustainable vaccines against babesiosis is currently hindered in part by the absence of definitive host correlates of protection. Despite that, studies in Babesia microti and Babesia bovis, major causative agents of human and bovine babesiosis, respectively, suggest that early activation of innate immune responses is crucial for vertebrates to survive acute infection. Trained immunity (TI) is defined as the development of memory in vertebrate innate immune cells, allowing more efficient responses to subsequent specific and non-specific challenges. Considering that Mycobacterium bovis bacillus Calmette-Guerin (BCG), a widely used anti-tuberculosis attenuated vaccine, induces strong TI pro-inflammatory responses, we hypothesize that BCG TI may protect vertebrates against acute babesiosis. This premise is supported by early investigations demonstrating that BCG inoculation protects mice against experimental B. microti infection and recent observations that BCG vaccination decreases the severity of malaria in children infected with Plasmodium falciparum, a Babesia-related parasite. We also discuss the potential use of TI in conjunction with recombinant BCG vaccines expressing Babesia immunogens. In conclusion, by concentrating on human and bovine babesiosis, herein we intend to raise awareness of BCG TI as a strategy to efficiently control Babesia infection.

Fundamental Tick Vaccinomic Approach to Evade Host Autoimmune Reaction.

Ticks are obligate hematophagous ectoparasites that infect domestic animals, humans, and wildlife. Ticks can transmit a wide range of pathogens (viruses, rickettsia, bacteria, parasites, etc.), and some of those are of zoonotic importance. Tick-borne diseases have a negative economic impact in several tropical and subtropical countries. With climate change, tick distribution and tick-associated pathogens have increased. Currently, tick control procedures have more environmental drawbacks and there are pitfalls in vaccination process. Since vaccinations have helped to prevent several diseases and infections, several vaccination trials are ongoing to control ticks and tick-borne pathogens. However, autoimmune reactions to vaccinations are reported as an adverse reaction since vaccines were used to protect against disease in humans and animals. The antibodies against the vaccine antigen might harm similar antigen in the host. Therefore, in this chapter, we attempt to shed light on the importance of raising awareness of possible adverse events associated with vaccinations and the methods that should be used to address this problem. In silico and lab work should be performed ahead of the vaccination process to evaluate the vaccine candidates and avoid the vaccination opposing consequences.

Babesia microti Immunoreactive Rhoptry-Associated Protein-1 Paralogs Are Ancestral Members of the Piroplasmid-Confined RAP-1 Family.

Babesia, Cytauxzoon and Theileria are tick-borne apicomplexan parasites of the order Piroplasmida, responsible for diseases in humans and animals. Members of the piroplasmid rhoptry-associated protein-1 (pRAP-1) family have a signature cysteine-rich domain and are important for parasite development. We propose that the closely linked B. microti genes annotated as BMR1_03g00947 and BMR1_03g00960 encode two paralogue pRAP-1-like proteins named BmIPA48 and Bm960. The two genes are tandemly arranged head to tail, highly expressed in blood stage parasites, syntenic to rap-1 genes of other piroplasmids, and share large portions of an almost identical ~225 bp sequence located in their 5' putative regulatory regions. BmIPA48 and Bm960 proteins contain a N-terminal signal peptide, share very low sequence identity (<13%) with pRAP-1 from other species, and harbor one or more transmembrane domains. Diversification of the piroplasmid-confined prap-1 family is characterized by amplification of genes, protein domains, and a high sequence polymorphism. This suggests a functional involvement of pRAP-1 at the parasite-host interface, possibly in parasite adhesion, attachment, and/or evasion of the host immune defenses. Both BmIPA48 and Bm960 are recognized by antibodies in sera from humans infected with B. microti and might be promising candidates for developing novel serodiagnosis and vaccines.

Rapid Detection of Equine Piroplasms Using Multiplex PCR and First Genetic Characterization of Theileria haneyi in Egypt.

Equine Piroplasmosis (EP) is an infectious disease caused by the hemoprotozoan parasites Theileria equi, Babesia caballi, and the recently identified species T. haneyi. Hereby, we used a multiplex PCR (mPCR) targeting the 18S rRNA gene of T. equi and B. caballi for the simultaneous detection of EP in Egyptian equids and examined the presence of T. haneyi infections in Egypt. Blood samples from 155 equids (79 horses and 76 donkeys) collected from different governorates of Egypt were examined by mPCR and PCR targeting T. hayeni. The mPCR method revealed a prevalence of T. equi of 20.3% in horses and of 13.1% in donkeys and a prevalence of B. caballi of 1.2% in horses. B. caballi was not detected in donkeys in the current study. The mPCR method also detected coinfections with both species (2.5% and 1.3% in horses and donkeys, respectively). Additionally, we report the presence of T. haneyi in Egypt for the first time in 53.1% of the horse and 38.1% of the donkey tested samples. Coinfection with T. haneyi and T. equi was found in 13.5% of the samples, while infection with the three EP species was found in 1.9% of the samples.

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.

The Structural Basis of Babesia orientalis Lactate Dehydrogenase.

Glycolytic enzymes play a crucial role in the anaerobic glycolysis of apicomplexan parasites for energy generation. Consequently, they are considered as potential targets for new drug development. Previous studies revealed that lactate dehydrogenase (LDH), a glycolytic enzyme, is a potential drug target in different parasites, such as Plasmodium, Toxoplasma, Cryptosporidium, and Piroplasma. Herein, in order to investigate the structural basis of LDH in Babesia spp., we determined the crystal structure of apo Babesia orientalis (Bo) LDH at 2.67-Å resolution in the space group P1. A five-peptide insertion appears in the active pocket loop of BoLDH to create a larger catalytic pocket, like other protozoa (except for Babesia microti LDH) and unlike its mammalian counterparts, and the absence of this extra insertion inactivates BoLDH. Without ligands, the apo BoLDH takes R-state (relaxed) with the active-site loop open. This feature is obviously different from that of allosteric LDHs in T-state (tense) with the active-site loop open. Compared with allosteric LDHs, the extra salt bridges and hydrogen bonds make the subunit interfaces of BoLDH more stable, and that results in the absence of T-state. Interestingly, BoLDH differs significantly from BmLDH, as it exhibits the ability to adapt quickly to the synthetic co-factor APAD+. In addition, the enzymatic activity of BoLDH was inhibited non-competitively by polyphenolic gossypol with a Ki value of 4.25 µM, indicating that BoLDH is sensitive to the inhibition of gossypol and possibly to its new derivative compounds. The current work provides the structural basis of BoLDH for the first time and suggests further investigation on the LDH structure of other Babesia spp. That knowledge would indeed facilitate the screening and designing of new LDH inhibitors to control the intracellular proliferation of Babesia spp.

Bovine Babesiosis in Turkey: Impact, Current Gaps, and Opportunities for Intervention.

Bovine babesiosis is a global tick-borne disease that causes important cattle losses and has potential zoonotic implications. The impact of bovine babesiosis in Turkey remains poorly characterized, but several Babesia spp., including B. bovis, B. bigemina, and B. divergens, among others and competent tick vectors, except Rhipicephalus microplus, have been recently identified in the country. Bovine babesiosis has been reported in all provinces but is more prevalent in central and highly humid areas in low and medium altitude regions of the country housing approximately 70% of the cattle population. Current control measures include acaricides and babesicidal drugs, but not live vaccines. Despite the perceived relevant impact of bovine babesiosis in Turkey, basic research programs focused on developing in vitro cultures of parasites, point-of-care diagnostic methods, vaccine development, "omics" analysis, and gene manipulation techniques of local Babesia strains are scarce. Additionally, no effective and coordinated control efforts managed by a central animal health authority have been established to date. Development of state-of-the-art research programs in bovine babesiosis to address current gaps in knowledge and implementation of long-term plans to control the disease will surely result in important economic, nutritional, and public health benefits for the country and the region.

Identification and antigenicity of the Babesia caballi spherical body protein 4 (SBP4).

BACKGROUND: The tick-borne intra-erythrocytic apicomplexan Babesia caballi is one of the etiological agents of equine babesiosis, an economically important disease of equids in most tropical and subtropical areas of the world. Discovering candidate antigens for improved diagnostic tools and vaccines remains needed for controlling equine babesiosis. This study describes the B. caballi sbp4 (Bcsbp4) gene and protein (BcSBP4) and analyzes its antigenicity in infected equids. METHODS: BLAST searches of an uncurated B. caballi assembly genome using the B. bovis SBP4 as a query were carried out, followed by PCR amplification and sequencing of a newly identified BcSBP4. Characterization of this novel gene and protein was performed by bioinformatics analysis, western blots, immunofluorescence (IFA) and an in vitro neutralization test using anti SBP4 peptide antibodies. Antigenicity of recombinant BcSBP4 (rBcSBP4) was tested with sera from field animals (n = 18) using an indirect ELISA (iELISA). RESULTS: Babesia caballi genome searches using B. bovis SBP4 as a query allowed identification of a novel gene termed Bcsbp4. The Bcsbp4 gene encodes for a protein of 30.58 kDa, which is fully conserved among B. caballi isolates from USA and Egypt. Bioinformatics analysis indicates that BcSBP4 contains a signal peptide and lacks additional transmembrane domains. Expression of BcSBP4 in blood stages of B. caballi was confirmed by western blot and IFA using antibodies against synthetic peptides representing putative B-cell epitopes of BcSBP4 predicted by in silico analysis. In vitro neutralization tests using anti-BcSBP4 peptide antibodies showed a marginal, but statistically significant inhibitory effect on the infectivity of B. caballi merozoites in horse red blood cells. Sera from eight B. caballi-infected equids, but none out of ten negative equid control sera, gave a positive signal in an rBcSBP4 based iELISA. CONCLUSIONS: The Bcsbp4 gene is expressed in B. caballi blood stages. The BcSBP4 protein is a potential candidate for developing a novel serological test that could detect B. caballi infection in equids in tropical and subtropical countries worldwide.

Transgenic Babesia bovis lacking 6-Cys sexual-stage genes as the foundation for non-transmissible live vaccines against bovine babesiosis.

Babesia bovis, a tick-borne apicomplexan parasite responsible for bovine babesiosis has a complex life cycle including sexual development in its Rhipicephalus microplus vector. Understanding the molecular mechanisms involved in sexual development is essential for developing future-generation transmission blocking vaccines (TBVs) and/or non-transmissible attenuated live vaccines. The widely conserved members of the 6-Cys gene family likely play roles in the development of sexual stages of B. bovis, and are candidates for developing novel TBV. The recently defined sexual markers 6-CysA and 6-CysB of B. bovis are strain-conserved and exclusively surface-expressed in tick-stage parasites. However, the high level of sequence identity among the 6-Cys A and 6-Cys B proteins (52% identity), together with similar 6-Cys domain distribution and sub-cellular localization, are suggestive of redundant function. We hypothesized that disruption of both 6-CysA and 6-CysB in B. bovis would result in unaltered ability of the parasite to invade and grow in red blood cells (RBCs), with concomitant loss of the transmission phenotype. Taking advantage of their contiguous genome localization, we generated a double gene-knockout system to disrupt a 3287 bp region encompassing both 6-CysA and 6-CysB genes using a single transfection plasmid. The resulting red-fluorescent ΔAΔB 6-Cys B. bovis transgenic parasite line was able to grow continuously in bovine RBCs in vitro at a similar rate to wild-type parasites, demonstrating that the 6-CysA and 6-CysB genes are not required for the development of blood-stage parasites. This novel gene manipulation approach will allow future experiments aimed at determining the tick-transmission phenotype of parasites lacking tick-stage genes. Parasites deficient in genes required for sexual reproduction could be the foundation for genetically-defined, non-transmissible live vaccines against bovine babesiosis. Developing a non-tick transmissible live vaccine based on attenuated parasites unable to express critical 6-Cys genes and including a molecular vaccine marker could help reduce the burden of bovine babesiosis globally.

Unravelling the cellular and molecular pathogenesis of bovine babesiosis: is the sky the limit?

The global impact of bovine babesiosis caused by the tick-borne apicomplexan parasites Babesia bovis, Babesia bigemina and Babesia divergens is vastly underappreciated. These parasites invade and multiply asexually in bovine red blood cells (RBCs), undergo sexual reproduction in their tick vectors (Rhipicephalus spp. for B. bovis and B. bigemina, and Ixodes ricinus for B. divergens) and have a trans-ovarial mode of transmission. Babesia parasites can cause acute and persistent infections to adult naïve cattle that can occur without evident clinical signs, but infections caused by B. bovis are associated with more severe disease and increased mortality, and are considered to be the most virulent agent of bovine babesiosis. In addition, babesiosis caused by B. divergens has an important zoonotic potential. The disease caused by B. bovis and B. bigemina can be controlled, at least in part, using therapeutic agents or vaccines comprising live-attenuated parasites, but these methods are limited in terms of their safety, ease of deployability and long-term efficacy, and improved control measures are urgently needed. In addition, expansion of tick habitats due to climate change and other rapidly changing environmental factors complicate efficient control of these parasites. While the ability to cause persistent infections facilitates transmission and persistence of the parasite in endemic regions, it also highlights their capacity to evade the host immune responses. Currently, the mechanisms of immune responses used by infected bovines to survive acute and chronic infections remain poorly understood, warranting further research. Similarly, molecular details on the processes leading to sexual reproduction and the development of tick-stage parasites are lacking, and such tick-specific molecules can be targets for control using alternative transmission blocking vaccines. In this review, we identify and examine key phases in the life-cycle of Babesia parasites, including dependence on a tick vector for transmission, sexual reproduction of the parasite in the midgut of the tick, parasite-dependent invasion and egression of bovine RBCs, the role of the spleen in the clearance of infected RBCs (IRBCs), and age-related disease resistance in cattle, as opportunities for developing improved control measures. The availability of integrated novel research approaches including "omics" (such as genomics, transcriptomics, and proteomics), gene modification, cytoadhesion assays, RBC invasion assays and methods for in vitro induction of sexual-stage parasites will accelerate our understanding of parasite vulnerabilities. Further, producing new knowledge on these vulnerabilities, as well as taking full advantage of existing knowledge, by filling important research gaps should result in the development of next-generation vaccines to control acute disease and parasite transmission. Creative and effective use of current and future technical and computational resources are needed, in the face of the numerous challenges imposed by these highly evolved parasites, for improving the control of this disease. Overall, bovine babesiosis is recognised as a global disease that imposes a serious burden on livestock production and human livelihood, but it largely remains a poorly controlled disease in many areas of the world. Recently, important progress has been made in our understanding of the basic biology and host-parasite interactions of Babesia parasites, yet a good deal of basic and translational research is still needed to achieve effective control of this important disease and to improve animal and human health.